Small and intermediate conductance, calcium-activated potassium channels and uses thereof

ABSTRACT

This invention relates to small and intermediate conductance, calcium-activated potassium channel proteins. More specifically, the invention relates to compositions and methods for making and detecting calcium-activated potassium channel proteins and the nucleic acids encoding calcium-activated potassium channel proteins. The invention also provides methods and compositions for assaying compounds which increase or decrease potassium ion flux through a calcium-activated potassium channel.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S.Ser. No. 60/026,451, filed on Sep. 11, 1997; U.S. Ser. No. 60/040,052,filed on Mar. 7, 1997; and U.S. Ser. No. 60/045,233, filed on Apr. 17,1997.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

[0002] This invention was made with United States Government supportunder Grant No. 1R01NS31872-01A1, awarded by the National Institutes ofHealth. The United States Government has certain rights in thisinvention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK.

[0003] Not Applicable

BACKGROUND OF THE INVENTION Field of the Invention

[0004] The present invention relates to compositions relating to, andmethods for identifying, small conductance (SK) and intermediateconductance (IK), calcium-activated potassium channels. The inventionfurther provides a method to assay for compounds that increase ordecrease potassium ion flux through calcium-activated potassiumchannels.

[0005] Calcium-activated potassium currents are found in a wide varietyof animal cells such as nervous, muscular, glandular or epithelialtissue and from the immune system. The channels regulating thesecurrents open and allow the escape of potassium as the internal calciumconcentration increases. This outward flow of potassium ions makes theinterior of the cell more negative, counteracting depolarizing voltagesapplied to the cell.

[0006] Two distinct classes of calcium-activated K⁺ channels (K_(ca)channels) have been described. Large conductance calcium-activated K⁺channels (BK channels) are gated by the concerted actions of internalcalcium ions and membrane potential, and have a unit conductance between100 and 220 pS. Small (SK) and intermediate (IK) conductancecalcium-activated K⁺ channels are gated solely by internal calcium ions,with a unit conductance of 2-20 and 20-85 pS, respectively, and are moresensitive to calcium than are BK channels (for review see Latorre etal., 1989, Ann Rev Phys, 51, 385-399.). In addition, each type of K_(Ca)channel shows a distinct pharmacological profile. All three classes arewidely expressed, and their activity hyperpolarizes the membranepotential. Members of the BK (Atkinson et al., 1991, Science, 253,551-555.; Adelman et al., 1992 Neuron, 9, 209-216.; Butler, 1993,Science, 261, 221-224) and SK (Kohler et al., 1996, Science, 273,1709-1714.) subfamilies have been cloned and expressed in heterologouscell types where they recapitulate the fundamental properties of theirnative counterparts.

[0007] In vertebrate neurons action potentials are followed by an afterhyperpolarization (AHP) that may persist for several seconds and haveprofound consequences for the firing pattern of the neuron. Alterationsin the AHP have been implicated in seizure activity (Alger et al., J.Physiol. 399:191-205 (1988)) and learning and memory (de Jonge et al.,Exp. Br. Res. 80:456-462 (1990)). The AHP is composed of two prominentcomponents, a fast component (fAHP) which mediates spike frequency atthe onset of a burst, and a subsequent slow component (sAHP) which isresponsible for spike-frequency adaptation (Nicoll, Science 241:545-551(1988)).

[0008] Each component of the AHP is kinetically distinct and is due toactivation of different calcium-activated potassium channels. Activationof large-conductance (100-200 picoSiemens (pS)), voltage- andcalcium-activated potassium channels (BK channels) underlies the fAHP(Lancaster et al, J. Physiol. 389:187-203 (1987); Viana et al., J.Neurophysiol. 69:2150-2163 (1993)) which develops rapidly (1-2 ms) anddecays within tens of milliseconds. The channels underlying the sAHP aresmall conductance, calcium activated, potassium channels (SK channels)which differ from BK channels, being more calcium-sensitive, are notvoltage-gated, and possessing a smaller unit conductance (Lancaster etal., J. Neurosci. 11:23-30 (1991); Sah, J. Neurophysiol. 74:1772-1776(1995)).

[0009] The fAHP and the sAHP also differ in their pharmacology. The fAHPis blocked by low concentrations of external tetraethylammonium (TEA)and charybdotoxin (CTX), in accord with the pharmacology of the BKchannels. Lancaster et al, J. Physiol. 389:187-203 (1987); Viana et al.,J. Neurophysiol. 69:2150-2163 (1993); Butler et al., Science 261:221-224(1993). In contrast, the sAHP is insensitive to CTX, but fall into twoclasses regarding sensitivity to the bee venom peptide toxin, apamin.For example, in hippocampal pyramidal neurons, the sAHP is insensitiveto apamin (Lancaster et al., J. Neurophysiol. 55:1268-1282 (1986)),while in hippocampal interneurons and vagal neurons it is blocked bynanomolar concentrations of the toxin (Sah, J. Neurophysiol.74:1772-1776 (1995); Zhang et al., J. Physiol. 488:661-672 (1995)).

[0010] In addition to its role in neuronal cells, non-voltage gated,apamin-sensitive potassium channels activated by submicromolarconcentrations of calcium have also been described from peripheral celltypes, including skeletal muscle (Blatz et al., Nature 323:718-720(1986)), gland cells (Tse et al., Science 255:462-464 (1992); Park, J.Physiol. 481:555-570 (1994)) and T-lymphocytes (Grissmer et al., J. Gen.Physiol. 99:63-84 (1992)).

[0011] For example, SK channels have been suggested to represent theapamin receptor found in muscle membrane of patients with myotonicmuscular dystrophy. Renaud et al., Nature 319:678-680 (1986)). Also,Grissmer et al. (J. Gen. Physiol. 99:63-84 (1992)) report that CTXinsensitive, apamin sensitive calcium-activated potassium channels wereidentified in a human leukemic T cell line and suggest thatcalcium-activated potassium channels play a supporting role duringT-cell activation by sustaining dynamic patterns of calcium signaling.And in many cells, SK channels are activated as a result ofneurotransmitter or hormone action. Haylett et al., in PotassiumChannels: Structure, Classification, Function and Therapeutic Potential(Cook, N. S., ed.), pp.71-95, John Wiley and Sons, 1990). Intermediatechannels play a role in the physiology of red blood cells.

[0012] Intermediate conductance, calcium activated potassium channelshave been previously described in the literature by theirelectrophysiology. The Gardos channel is opened by submicromolarconcentrations of internal calcium and has a rectifying unitconductance, ranging from 50 pS at −120 mV to 13 pS at 120 mV(symmetrical 120 mM K⁺; Christophersen, 1991, J. Membrane Biol., 119,75-83.). It is blocked by charybdotoxin (CTX) but not the structurallyrelated peptide iberiotoxin (IBX), both of which block BK channels(Brugnara et al., 1995a, J. Membr. Biol., 147, 71-82). Apamin, a potentblocker of certain native (Vincent et al., 1975, J. Biochem., 14, 2521.;Blatz and Magleby, 1986, Nature, 323, 718-720.) and cloned SK channelsdo not block IK channels (de-Allie et al.,1996, Br. J. Pharm.,117,479-487). The Gardos channel is also blocked by some imidazolecompounds, such as clotrimazole, but not ketoconazole (Brugnara et al.,1993, J. Clin. Invest., 92,520-526). The electrophysiological andpharmacological properties of the Gardos channel show that it belongs tothe IK subfamily of this invention.

[0013] IK channels have been described in a variety of other cell types.Principle cells of the rat cortical collecting duct segregate differentclasses of K⁺ channels to the luminal and basolateral membranes. IKchannels are present in the basolateral membrane where they promote therecirculation of K⁺ across this membrane, elevating the activity of theNa⁺+K⁺-ATPase and thereby Na⁺ reabsorption into the blood (Hirsch andSchlatter, 1995, Pflügers Arch.-Eur. J. Physiol., 449, 338-344.) IKchannels have also been implicated in the microvasculature of the kidneywhere they may be responsible for the vasodilatory effects of bradykinin(Rapacon et al., 1996). In brain capillary endothelial cells, IKchannels are activated by endothelin, produced by neurons and glia,shunting excess K⁺ into the blood (Renterghem et al., 1995, J.Neurochem., 65, 1274-1281). Neutrophil granulocytes, mobile phagocyticcells which defend against microbial invaders, undergo a largedepolarization subsequent to agonist stimulation, and IK channels havebeen implicated in repolarizing the stimulated granulocyte (Varnai etal., 1993, J. Physiol., 472, 373-390.). IK channels have also beenidentified in both resting and activated human T-lymphocytes. Grissmeret al. 1993, J. Gen. Physiol. 102,601-630 reported that IK channels wereblocked by low nanomolar concentrations of charybdotoxin, showed littleor no voltage dependence, and were insensitive to apamin. This channelhas also been identified in human erythrocytes, where it plays animportant role in intracellular volume homeostasis (Joiner, C. H., 1993,Am. J. Physiol. 264: C251-270 and in smooth muscle (Van Renterghem, C.et al. 1996, J. Neurochemistry 65,1274-1281.

[0014] Thus, it appears that SK and IK channels comprise a subfamily ofcalcium-activated potassium channels which play key physiological rolesin many cell types. Accordingly, given the key role of SK and IKchannels in a wide variety of physiological functions, what is needed inthe art is the identification of novel SK and IK channel proteins andthe nucleic acids encoding them. Additionally, what is needed aremethods of identifying compounds which increase or decrease SK and IKchannel currents for their use in the treatment or regulation of:learning and memory disorders, seizures, myotonic dystrophies, immuneresponses, and neurotransmitter or hormone secretions. The presentinvention provides these and other advantages.

SUMMARY OF THE INVENTION

[0015] In a first broad context, this invention provides for novelproteins and their corresponding nucleic acids where the proteins aredefined as monomers of calcium activated potassium ion channels. Themonomers have a molecular weight of between 40 and 80 kDa and have unitsof conductance of between 2 and 80 pS when the monomer is in thepolymeric form as expressed in Xenopus oocytes. In addition, the monomerspecifically binds to antibodies generated against SEQ ID NO:30 or 42.

[0016] In another aspect, the present invention relates to an isolatednucleic acid encoding at least 15 contiguous amino acids of acalcium-activated potassium channel protein. The SK channel protein hasa sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47 and conservatively modifiedvariants of SEQ ID NOS: 1, 2, 3, 4, 19, 20, 32, 43 or 47.

[0017] In some embodiments, the isolated nucleic acid encodes acalcium-activated potassium channel protein having a conductance of atleast 2 pS when expressed in a Xeuopus oocyte, a molecular weight ofbetween 40 and 100 kilodaltons (kd), and selectively hybridizes, understringent hybridization conditions, with SK or IK encoding nucleic acidSuch as SEQ ID NO:13 in a human genomic library or SEQ ID NO:14 in a ratgenomic library. In other embodiments, the isolated nucleic acidencoding the calcium-activated potassium channel protein encodes aprotein having a sequence selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47. In preferred embodimentsthe nucleic acid has a sequence selected from the group consisting of:SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:31, SEQ ID NO:44, and SEQ ID NO:48.

[0018] In another aspect, the present invention relates to an isolatedcalcium-activated potassium channel protein having at least 15contiguous amino acids of a sequence selected from the group consistingof: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47, andconservatively modified variants of SEQ ID NOS: 1, 2, 3, 4, 19, 20, 32,43, or 47, wherein the variant specifically reacts, underimmunologically reactive conditions, with an antibody reactive to aprotein selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:30, SEQID NO:32, SEQ ID NO:43, and SEQ ID NO:47.

[0019] In a broad embodiment, the calcium-activated potassium channelprotein is defined as having a conductance of at least 2 pS and amolecular weight of between 40 and 100 Kd. In other embodiments, thecalcium-activated potassium channel protein has an amino acid sequenceselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ IDNO:43, and SEQ ID NO:47.

[0020] In another aspect, the present invention is directed to anantibody specifically reactive, under immunologically reactiveconditions, to a calcium-activated potassium channel protein, where theprotein has a sequence selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47. In preferred embodiments,the antibody is limited to a monoclonal antibody.

[0021] In yet another aspect, the present invention relates to anexpression vector comprising a nucleic acid encoding a monomer of acalcium-activated potassium channel where the monomer has a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:1, SEQ ID NO:20, SEQ ID NO:32, SEQ IDNO:43, and SEQ ID NO:47, and conservatively modified variants of SEQ IDNOS: 1, 2, 3, 4, 19, 20, 32, 43, or 47 wherein the modified variant is aprotein having a conductance of at least 2 pS when expressed in aXenopus oocyte, a molecular weight of between 40 and 100 kd, andspecifically reacts, under immunologically reactive conditions, with anantibody reactive to a full-length protein selected from the groupconsisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47.

[0022] In another aspect, the present invention relates to a host celltransfected with a vector comprising a nucleic acid encoding a monomerof a calcium-activated potassium channel protein where the protein has asequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, SEQID NO:47, and conservatively modified variants of SEQ ID NO:1, 2, 3, 4,19, 20, 32, 43 or 47 wherein the modified variant is a protein having aconductance of at least 2 pS when expressed in a Xenopus oocyte, amolecular weight of between 40 and 100 Kd, and specifically reacts,under immunologically reactive conditions, with an antibody reactive toa full-length protein selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47. Typically, the host cell iscultured under conditions permitting expression of the nucleic acidencoding the calcium-activated potassium channel protein.

[0023] In yet a further aspect, the present invention relates to anisolated nucleic acid sequence of at least 15 nucleotides in lengthwhich specifically hybridizes, under stringent conditions, to a nucleicacid encoding a calcium-activated potassium channel protein, where theprotein is selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47.

[0024] In an additional aspect, the present invention is directed to amethod for detecting the presence of a calcium-activated potassiumchannel protein in a biological sample. The method comprises contactingthe biological sample with an antibody, wherein the antibodyspecifically reacts, under immunologically reactive conditions, to ancalcium-activated potassium channel protein having a sequence selectedfrom the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQID NO:47 and allowing the antibody to bind to the protein underimmunologically reactive conditions, wherein detection of the boundantibody indicates the presence of the channel protein.

[0025] In yet another aspect, the present invention provides a methodfor detecting the presence, in a biological sample, of a nucleic acidsequence encoding a calcium-activated potassium channel protein of atleast 25 amino acias in length. The method comprises contacting thebiological sample, under stringent hybridization conditions, with anucleic acid probe comprising a nucleic acid segment that selectivelyhybridizes to a nucleic acid encoding the channel protein having asequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQID NO:43, and SEQ ID NO:47; allowing the nucleic acid encoding thechannel protein to selectively hybridize to the probe to form ahybridization complex, wherein detection of the hybridization complex isan indication of the presence of the nucleic acid sequence in thesample. In some embodiments, the hybridization conditions are moderatestringency hybridization conditions. In another embodiment, the calciumactivated channel protein is at least 400 amino acid residues in lengthand when expressed in oocytes has a conductance of at least 2 pS. In afurther embodiment, the nucleic acid probes comprises at least 250contiguous nucleotides encoding a subsequence within the small orintermediate calcium-activated potassium channel protein core region.

[0026] In a further aspect, the present invention relates to an isolatedcalcium-activated potassium channel encoded by a nucleic acid amplifiedby primers which selectively hybridize, under stringent hybridizationconditions, to the same nucleic acid sequence as primers selected fromthe group consisting of: for hSK1, SEQ ID NO:5 and SEQ ID NO:6; for rSK2SEQ ID NO:7 and SEQ ID NO:8; for endogenous rSK3, SEQ ID NO:9 and SEQ IDNO:10; for rSK1, SEQ ID NO:11 and SEQ ID NO:12; for hSK2, SEQ ID NO:23and SEQ ID NO:24; for hSK3, SEQ ID NO:25 and SEQ ID NO:26; and for hIKthe following primer pairs will amplify a probe that is selective foridentifying hIK1 from a human genomic or cDNA library: 5′GCCGTGCGTGCAGGATTTAGG 3′ (SEQ ID NO:34) and 5′ CCAGAGGCCAAGCGTGAGGCC 3′(SEQ ID NO:35) yielding a probe of about 270 bases or 5′TCCAAGATGCACATGATCCTG 3′ (SEQ ID NO:36) and 5′ GGACTGCTGGCTGGGTTCTGG 3′(SEQ ID NO:37) yielding a probe of about 165 bases. For amplification ofa full length hIK1 either of the following two primer pairs will work:5′ ATGGGCGGGGATCTGGTGCTTG 3′ (SEQ ID NO:38) and 5′CTACTTGGACTGCTGGCTGGGTTC 3′ (SEQ ID NO:39) or 540ATGGGCGGGGATCTGGTGCTTGG 3′ (includes codon of initiator methionine) (SEQID NO:40) and 5′ GGGTCCAGCTACTTGGACTGCTG 3′ (includes stop codon for endof translation) (SEQ ID NO:41).

[0027] In yet another aspect, the present invention relates to a methodof identifying a compound which increases or decreases the potassium ionflux through a small or intermediate conductance, calcium-activatedpotassium channel, with the provisio that the compound is notclotrimizole. The method comprises the steps of contacting the compoundwith a eukaryotic host cell in which has been expressed a nucleic acidencoding a calcium-activated potassium channel having a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ IDNO:43, SEQ ID NO:47 and conservatively modified variants thereof,wherein said conservatively modified variant specifically binds toantibodies specifically reactive with an antigen having an amino acidsequence selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47, have a conductance of at least 2pS, and a molecular weight between 40 and 100 kilodaltons; anddetermining the increased or decreased flux of potassium ions throughsaid channel. In preferred embodiments, the increased or decreased fluxof potassium ions is determined by measuring the electrical current orflux of ions, or indirectly the change in voltage induced by the changein current or flux of ions, across the cell membrane of said eukaryotichost cell. In a particularly preferred embodiment, the channel proteinhas a sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47. In another preferred embodiment,the channel protein is recombinant.

[0028] In a further aspect, the present invention relates to an isolatedeukaryotic nucleic acid encoding a calcium-activated potassium channelprotein of at least 400 amino acid residues in length, wherein thecalcium-activated channel protein comprises an amino acid sequencehaving at least 55 to 60% similarity over the length of a core region ofa protein selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47 and wherein the channel proteinhas a conductance of at least 2 pS. In some embodiments, the presentinvention is directed to the protein encoded by the aforementionedisolated eukaryotic nucleic acid. In other embodiments, the isolatednucleic acid encoding the calcium-activated channel protein has at least85% sequence similarity over a comparison window of 20 contiguous aminoacid residues within the core region.

[0029] In a further aspect, the present invention is directed to avector comprising an isolated eukaryotic nucleic acid encoding acalcium-activated potassium channel protein of at least 400 amino acidresidues in length, wherein the channel protein comprises an amino acidsequence having at least 55% similarity over the length of a core-regionof a protein selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47, and wherein the channel proteinhas a conductance of at least 2 pS. Typically, the vector is transfectedinto a host cell which is cultured under conditions permittingexpression of the isolated eukaryotic nucleic acid encoding the channelprotein.

[0030] In a further aspect, present invention is directed to a method ofidentifying a compound that increases or decreases the potassium ionflux through a calcium-activated potassium channel. The methodscomprises the steps of contacting the compound with a eukaryotic hostcell in which has been expressed a calcium-activated potassium channelprotein of at least 400 amino acid residues in length, wherein thechannel protein has an amino acid sequence having at least 55%similarity over the length of a core region of a protein selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ IDNO:47, and wherein the channel protein has a conductance of at least 2pS; and determining the increased or decreased flux of potassium ionsthrough the channel protein. In some embodiments the increased ordecreased flux of potassium ions is determined by measuring theelectrical current across the cell membrane of the eukaryotic host cell.

[0031] In another aspect, the present invention provides in a computersystem a method of screening for mutations of SK and IK genes, themethod comprising the steps of: (i) receiving input of a first nucleicacid sequence encoding a calcium-activated channel protein having asequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4,19, 20, 32, 43, 47 and conservatively modified versions thereof; (ii)comparing the first nucleic acid sequence with a second nucleic acidsequence having substantial identity to the first nucleic acid sequence;and (iii) identifying nucleotide differences between the first andsecond nucleic acid sequences. In one embodiment, the second nucleicacid sequence is associated with a disease state.

[0032] In another aspect, the invention provides in a computer system, amethod for identifying a three-dimensional structure of SK and IKproteins, the method comprising the steps of: (i) receiving input of anamino acid sequence of a calcium-activated channel protein or anucleotide sequence of a gene encoding the protein, the protein havingan amino acid sequence selected from the group consisting of SEQ IDNOS:1, 2, 3, 4, 19, 20, 32, 43, 47, and conservatively modified versionsthereof; and (ii) generating a three-dimensional structure of theprotein encoded by the amino acid sequence. In one embodiment, the aminoacid sequence is a primary structure and-the generating step-includesthe steps of forming a secondary structure from the primary structureusing energy terms encoded by the primary structure and forming atertiary structure from the secondary structure using energy termsencoded by said secondary structure. In another embodiment, thegenerating step includes the step of forming a quaternary structure fromthe tertiary structure using anisotropy terms encoded by the tertiarystructure. In another embodiment, the method further comprises the stepof identifying regions of the three-dimensional structure of the proteinthat bind to ligands and using the regions to identify ligands that bindto the protein.

BRIEF DESCRIPTION OF THE DRAWINGS DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention provides novel isolated, small conductance,calcium-activated potassium (SK) channels, intermediate conductance,calcium-activated potassium (IK) channels (collectively,“calcium-activated potassium channels”), and isolated nucleic acidsencoding SK and IK channels (i.e., SK and IK channel nucleic acids). Thedistribution, function, and pharmacology define these new classes ofchannels as SK or IK channels.

[0034] Expression of isolated SK or IK channel protein encoding nucleicacids in a host cell provides a composition which can be used toidentify compounds that increase or decrease potassium ion flux throughsmall conductance, calcium-activated potassium (SK) channels orintermediate conductance, calcium-activated potassium (IK) channels,respectively. Since SK channels underlie the slow component of theafterhyperpolarization (sAHP) of neurons, alteration of neuronal sAHPprovides a means to inhibit epileptic seizures or modulate learning ormemory disorders.

[0035] Calcium activated, SK channels are also implicated in T-cellactivation. Thus, increasing or decreasing SK channel currents providesa means to inhibit or potentiate the immune response. Moreover, SKchannels are associated with hormone and neurotransmitter secretions.Accordingly, altering SK channel currents provides a means to regulatecellular or glandular secretions and thereby treat imbalances thereof.

[0036] Calcium activated intermediate channels (IK) are also believed toplay an important physiological role particularly in peripheral tissues.For example, intermediate channels are reported in red blood cells, and,in part, contribute to cell dehydration, a process that is exacerbatedin sickle cell anemia.

[0037] The invention also relates to subsequences of isolated smallconductance and intermediate conductance, calcium-activated potassiumchannels and for isolated nucleic acids encoding SK and IK channelproteins. Isolated nucleic acids coding for SK or IK channel proteinsprovide utility as probes for identification of aberrant transcriptionproducts or increased or decreased transcription levels of genes codingfor SK or IK channels. Assaying for increased or decreased transcriptioncan be used in drug screening protocols. Likewise, SK or IK channelproteins can be used as immunogens to generate antibodies for use inimmunodiagnostic assays of increased or decreased expression ofcalcium-activated potassium channels in drug screening assays.

Definitions

[0038] Units, prefixes, and symbols may be denoted in their SI acceptedform. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation; amino acid sequences are written left toright in amino to carboxy orientation, respectively. Numeric ranges areinclusive of the numbers defining the range. The terms defined below aremore fully defined by reference to the specification as a whole.

[0039] The terms “nucleic acid” “probe”, or “primer” includes referenceto a deoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues of natural nucleotides that hybridize to nucleic acids inmanner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence includes the perfectcomplementary sequence thereof. Eukaryotic nucleic acids are nucleicacids from eukaryotic cells, preferably cells of multicellulareukaryotes.

[0040] The term “recombinant” when used with reference to a cell, orprotein, nucleic acid, or vector, includes reference to a cell, protein,or nucleic acid, or vector, that has been modified by the introductionof a heterologous nucleic acid or the alteration of a native nucleicacid to a form not native to that cell, or that the cell is derived froma cell so modified. Thus, for example, recombinant cells express genesand proteins that are not found within the native (non-recombinant) formof the cell or express native genes that are otherwise abnormallyexpressed, under expressed or not expressed at all.

[0041] The term “subsequence” in the context of a referenced nucleicacid sequence includes reference to a contiguous sequence from thenucleic acid having fewer nucleotides in length than the referencednucleic acid. In the context of a referenced protein, polypeptide, orpeptide sequence (collectively, “protein”), “subsequence” refers to acontiguous sequence from the referenced protein having fewer amino acidsthan the referenced protein.

[0042] The terms “identical” or “sequence identity” in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of I and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

[0043] A “comparison window”, as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison may be conducted by the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482; by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48: 443; by the search for similarity method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerizedimplementations of these algorithms (including, but not limited toCLUSTAL in the PC/Gene program by Intelligenetics, Mountain View,Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group (GCG), 575 Science Dr.,Madison, Wis., USA); the CLUSTAL program is well described by Higginsand Sharp (1988) Gene, 73: 237-244 and Higgins and Sharp (1989) CABIOS5: 151-153; Corpet, et al. (1988) Nucleic Acids Research 16, 10881-90;Huang, et al. (1992) Computer Applications in the Biosciences 8, 155-65,and Pearson, et al. (1994) Methods in Molecular Biology 24, 307-31.Alignment is also often-performed by inspection and manual alignment.

[0044] The terms “substantial identity” or “similarity” ofpolynucleotide sequences means that a polynucleotide comprises asequence that has at least 60% sequence identity, preferably at least80%, more preferably at least 90% and most preferably at least 95%,compared to a reference sequence using the programs described above(preferably BLAST) using standard parameters. One indication that twonucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.

[0045] Another indication that two nucleic acid sequences havesubstantially identity is that the two molecules hybridize to each otherunder “moderate stringency hybridization conditions” (or “moderateconditions”). Exemplary “moderate stringency hybridization conditions”include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDSat 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is atleast twice background. Those of ordinary skill will readily recognizethat alternative hybridization and wash conditions can be utilized toprovide conditions of similar stringency. Nucleic acids which do nothybridize to each other under moderate stringency hybridizationconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

[0046] The terms “substantial identity” or “similarity” in the contextof a peptide indicates that a peptide comprises a sequence with at least60% sequence identity to a reference sequence, usually at least 70%,preferably 80%, more preferably 85%, most preferably at least 90% or 95%sequence identity to the reference sequence over a specified comparisonwindow. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443.An indication that two peptide sequences are substantially identical isthat one peptide is immunologically reactive with antibodies raisedagainst the second peptide. Thus, a peptide is substantially identicalto a second peptide, for example, where the two peptides differ only bya conservative substitution. Generally, similarity is determined using acomparison window having a length of any number from 20 contiguouspositions to the number of residues in the full-length core regionsequence (i.e., the region of optimal alignment with rSK2 from aminoacid residue 135 to 462), where the comparison window is within the coresequence.

[0047] The terms “oligonucleotide” or “polynucleotide” probes includereference to both double stranded and single stranded DNA or RNA. Theterms also refer to synthetically or recombinantly derived sequencesessentially free of non-nucleic acid contamination.

[0048] As used herein, “contact” or “contacting” means to place indirect physical association.

[0049] “Biological sample” as used herein is a sample of biologicaltissue or fluid that contains an IK and/or SK channel protein or nucleicacid encoding the corresponding IK and/or SK channel protein. Suchsamples include, but are not limited to, sputum, amniotic fluid, blood,blood cells (e.g., white cells), or tissue. Biological samples may alsoinclude sections of tissues such as frozen sections taken forhistological purposes. Examples of biological samples include a cellsample from nervous, muscular, glandular or epithelial tissue or fromthe immune system (e.g., T cells). A biological sample is typicallyobtained from a eukaryotic organism, preferably a multicellulareukaryotes such as insect, protozoa, birds, fish, reptiles, andpreferably a mammal such as rat, mice, cow, dog, guinea pig, or rabbit,and most preferably a primate such as macaques, chimpanzees, or humans.

[0050] The term “antibody” also includes antigen binding forms ofantibodies (e.g., Fab, F(ab)₂). The term “antibody” refers to apolypeptide substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof which specifically bind andrecognize an analyte (antigen). The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as the myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively.

[0051] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

[0052] Antibodies exist e.g., as intact immunoglobulins or as a numberof well characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see, Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. 1993). While various antibody fragmentsare defined in terms of the digestion of an intact antibody, one ofskill will appreciate that such fragments may be synthesized de novoeither chemically or by utilizing recombinant DNA methodology. Thus, theterm antibody, as used herein, also includes antibody fragments such assingle chain Fv, chimeric antibodies (i.e., comprising constant andvariable regions from different species), humanized antibodies (i.e.,comprising a complementarity determining region (CDR) from a non-humansource) and heteroconjugate antibodies (e.g., bispecific antibodies).

[0053] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0054] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

[0055] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art.

[0056] The following six groups each contain amino acids that areconservative substitutions for one another:

[0057] 1)Alanine (A), Serine (S), Threonine (T);

[0058] 2)Aspartic acid (D), Glutamic acid (E);

[0059] 3)Asparagine (N), Glutamine (Q);

[0060] 4)Arginine (R), Lysine (K);

[0061] 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0062] 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0063] See also, Creighton (1984) Proteins W. H. Freeman and Company.

[0064] The terms “biologically pure” or “isolated” refer to materialwhich is substantially or essentially free from components whichnormally accompany or interact with it as found in its naturallyoccurring environment. The isolated material optionally comprisesmaterial not found with the material in its natural environment.

[0065] The phrase “encodes a protein which could be encoded by a nucleicacid that selectively hybridizes under moderate stringency hybridizationconditions to a sequence selected from the group consisting of:” in thecontext of nucleic acids refers to those nucleic acids encodingnaturally occurring proteins or derivatives of natural proteins, butwhich are deliberately modified or engineered to no longer hybridize tothe protein of natural origin under the stated conditions.

[0066] An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements which permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed, and a promoter.

[0067] The phrase “functional effects” in the context of assays fortesting compounds affecting the channel includes the determination ofany parameter that is indirectly or directly under the influence of thechannel. It includes changes in ion flux and membrane potential but alsoincludes other physiologic effects such increases or decreases oftranscription or hormnone release.

[0068] By “selectively hybridizing” or “selective hybridization” or“selectively hybridizes” is meant hybridization, under stringenthybridization conditions, of a nucleic acid sequence to a specifiednucleic acid target sequence to a detectably greater degree than itshybridization to non-target nucleic acid sequences and/or to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences have at least 80% sequence identity, preferably90% sequence identity, and most preferably 100% sequence-identity (i.e.,complementary) with each other. “Percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

[0069] The terms “stringent conditions” or “stringent hybridizationconditions” refer to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The T_(m) isthe temperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of30% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 2×SSC at 50° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C.

[0070] “Stringent hybridization conditions” or “stringent conditions” inthe context of nucleic acid hybridization assay formats are sequencedependent, and are different under different environmental parameters.An extensive guide to the hybridization of nucleic acids is found inTijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.

[0071] By “hybridization complex” is meant a duplex nucleic acidsequence formed by selective hybridization of two single-strandednucleic acid sequences with each other.

[0072] By “host cell” is meant a cell which contains an expressionvector and supports the replication or expression of the expressionvector. Host cells may be prokaryotic cells such as E. coli, oreukaryotic cells such as yeast, insect, amphibian, or mammalian cells.

[0073] By “conductance” is meant electrical conductance. Electricalconductance is conveniently measured in Siemens (1/ohm=mho). Unitaryconductance is determined by measuring single channel currents using apatch clamp protocol under conditions set forth in Example 6 (i.e., inan oocyte) using a symmetrical potassium ion concentration of 120 mM.See generally, Hille, B., Ionic Channels of Excitable Membranes, 2nded., Sinauer Assoc., Sunderland, Mass. In the context of the presentinvention, “conductance” refers to the unitary electrical conductance ofa single homomeric protein of the referenced SK or IK channel protein.

[0074] By “when expressed in an oocyte leads to formation of an SKchannel” includes reference to expression of a referenced SK protein inwhich a plurality of the referenced SK proteins are assembled to form,by themselves or in conjunction with other endogenous Xenopus oocytemolecules, an SK channel. Expression within a Xenopus oocyte isdisclosed in the Examples provided herein, e.g., Example 3.

[0075] By “when expressed in an oocyte leads to formation of acalcium-activated potassium channel” includes reference to expression ofa referenced IK and/or SK protein in which a plurality of the referencedIK and/or SK proteins are assembled to form, by themselves or inconjunction with other endogenous Xenopus oocyte molecules, acalcium-activated potassium channel. Expression within a Xenopus oocyteis disclosed in the Examples provided herein, e.g., Example 3.

[0076] By “immunologically reactive conditions” is meant conditionswhich allow an antibody, generated to a particular epitope, to bind tothat epitope to a detectably greater degree than the antibody binds tosubstantially all other epitopes. Immunologically reactive conditionsare dependent upon the format of the antibody binding reaction andtypically are those utilized in immunoassay protocols. See Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, for a description of immunoassay formats andconditions.

[0077] By “antibody reactive to a protein” is meant the protein is“specifically immunoreactive with an antibody.”

[0078] The phrase “specifically immunoreactive with an antibody”, or“specifically binds to an antibody” when referring to a protein orpeptide, refers to a binding reaction between an antibody and a proteinhaving an epitope recognized by the antigen binding site of theantibody. This binding reaction is determinative of the presence of aprotein having the recognized epitope amongst the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aprotein having the recognized epitope and bind, if at all, to adetectably lesser degree to other proteins lacking the epitope which arepresent in the sample.

[0079] Specific binding to an antibody under such conditions may requirean antibody that is selected for its specificity for a particularprotein. For example, antibodies raised to the calcium activatedpotassium channel protein with the amino acid sequence depicted in SEQID NO:1, 2, 3, 4, 19, 20, 30, 32, 43, and 47 can be selected from toobtain antibodies specifically immunoreactive with small and/orintermediate calcium activated potassium channel proteins and not withother proteins. The proteins used as immunogens can be in nativeconformation or denatured so as to provide a linear epitope.

[0080] A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

[0081] By “transfected” is meant the introduction of a nucleic acid intoa eukaryotic cell where the nucleic acid may be incorporated into thegenome of the cell (i.e., chromosome, plasmid, or mitochondrial DNA),converted into an autonomous replicon, or transiently expressed (e.g.,transfected mRNA). The transfection can be in vivo or ex vivo. “Ex vivo”means outside the body of the organism from which a cell or cells isobtained or from which a cell line is isolated. Ex vivo transfection ispreferably followed by re-infusion of the cells back into the organism.In contrast, by “in vivo” is meant within the body of the organism fromwhich the cell was obtained or from which a cell line is isolated.

[0082] By “antigen” is meant a substance to which an antibody can begenerated and to which the antibody is specifically immunoreactive with.An antibody immunologically reactive with a particular antigen can begenerated in vivo or by recombinant methods such as selection oflibraries of recombinant antibodies in phage or similar vectors. See,e.g., Huse et al. (1989) Science 246:1275-1281; and Ward, et al. (1989)Nature 341:544-546; and Vaughan et al. (1996) Nature Biotechnology,14:309-314.

[0083] By “encoding” or “encoded”, with respect to a specified nucleicacid, is meant comprising the information for translation into thespecified protein. The information is specified by the use of codons.Typically, the amino acid sequence is encoded by the nucleic acid usingthe “universal” genetic code. However, variants of the universal code,such as is present in some plant, animal, and fungal mitochondria, thebacterium Mycoplasma capricolum (Proc. Natl. Acad. Sci., 82:2306-2309(1985), or the ciliate Macronucleus, may be used when the nucleic acidis expressed using these organisms.

[0084] By “contiguous amino acids from” in the context of a specifiednumber of amino acid residues from a specified sequence, is meant asequence of amino acids of the specified number from within thespecified reference sequence which has the identical order of aminoacids each of which is directly adjacent to the same amino acids as inthe reference sequence.

[0085] By “small conductance, calcium activated potassium channel” or“SK channel” is meant a membrane channel which is not voltage-gated,activated by calcium from about 30 nM to 10 μM, and has a unitaryconductance of from about 2 to 60 pS, often 2 to 25 pS, when measuredunder a symmetrical potassium concentration of 120 mM using theconditions specified in Example 6. An SK channel comprises multiple SKchannel proteins as subunits, typically four SK channel proteins (e.g.,full length or substantially full length SK channel proteins).

[0086] By “small conductance, calcium-activated channel protein” or “SKchannel protein” is meant a peptide of at least 10 contiguous aminoacids in length from an amino acid sequence which makes up an SKchannel. These proteins, when full length, serve as monomers of the SKchannel. Thus, an SK channel protein can have the functionalcharacteristics to form a heteromeric or homomeric protein with thefunctional characteristics of an SK channel, or be a peptide fragmentthereof. For example, both N-terminal extended rsk3 (SEQ ID NO:43 andtruncated rsk3 (SEQ ID NO:3) demonstrate virtually identical functionalcharacteristics.

[0087] By “intermediate conductance, calcium-activated potassiumchannel” or “IK channel” is meant a membrane channel which is notvoltage-gated, activated by calcium from about 30 nM to 10 μM, and hasin its broadest context a unitary inward conductance of from about 20 to80 pS, but more likely 30 to 70 pS, 40 to 60 pS, or most preferablyabout 35 to 40 pS when measured under a symmetrical potassiumconcentration of 120 mM using the conditions specified in Example 6. AnIK channel comprises multiple IK channel proteins as subunits, typicallyfour IK channel proteins (e.g., full length or substantially full lengthIK channel proteins).

[0088] By “intermediate conductance, calcium-activated channel protein”or “IK channel protein” is meant a peptide of at least 10 contiguousamino acids in length from an amino acid sequence which makes up an IKchannel. These proteins, when full length, serve as monomers of the IKchannel. Thus, an IK channel protein can have the functionalcharacteristics to form a heteromeric or homomeric protein with thefunctional characteristics of an IK channel, or be a peptide fragmentthereof.

[0089] By “calcium-activated potassium channel” means a smallconductance, calcium-activated potassium (SK) channel, and anintermediate conductance, calcium-activated potassium (IK) channel.

[0090] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

[0091] By “specifically reacts” or “specifically reactive” is meant areaction of the specificity exhibited by that between an antibody and aprotein which “specifically binds” with that antibody.

[0092] By “human genomic library” is meant a collection of isolated DNAmolecules which substantially represent the entire genome of a human.Construction of genomic libraries is taught in standard molecularbiology references such as Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif. (Berger); Sambrook et al. (1989) Molecular Cloning—ALaboratory Manual (2nd ed.) Vol. 1-3; and Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1994 Supplement) (Ausubel).

[0093] By “amplified” is meant the construction of multiple copies of anucleic acid sequence or multiple copies complementary to the nucleicacid sequence using at least one of the nucleic acid sequences as atemplate. Amplification systems include the polymerase chain reaction(PCR) system, ligase chain reaction (LCR) system, nucleic acid sequencebased amplification (NASBA, Cangene, Mississauga, Ontario), Q-BetaReplicase systems, transcription-based amplification system (TAS), andstrand displacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, Ed. D. H. Persing et al.,American Society for Microbiology, Washington, D.C.

[0094] The term “residue” or “amino acid residue” or “amino acid” asused herein refers to an amino acid that is incorporated into a protein,polypeptide, or peptide (collectively “peptide”). The amino acid may bea naturally occurring amino acid and, unless otherwise limited, mayencompass known analogs of natural amino acids that can function in asimilar manner as naturally occurring amino acids.

[0095] By “segment of nucleic acid” is meant a nucleic acid sequence ofany one of from 15 to about 1500 nucleotides, or nucleotide analogs, inlength or concatamers of such sequence.

[0096] By “determining the functional effect” is meant examining theeffect of a compound that increases or decreases potassium ion flux on acell or cell membrane in terms of cell and cell membrane function.Preferably, the term refers to the functional effect of the compound onSK and IK channel activity, e.g., changes in conductance, voltage gatingand the like.

Small and Intermediate Conductance, Calcium-Activated Potassium ChannelProteins

[0097] The present invention provides intermediate conductance,calcium-activated (IK) potassium channel proteins, and smallconductance, calcium-activated (SK) channel proteins (collectively,“calcium-activated potassium channels”). The isolated small conductance,calcium-activated (SK) channel proteins of the present inventioncomprise at least N amino acids from any one of the sequences selectedfrom the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:43, and SEQ ID NO:47, andconservatively modified variants thereof, where N is any one of theintegers selected from the group consisting of from 10 to 600 and thesequence is unique to the protein of origin.

[0098] Similarly, the isolated intermediate conductance,calcium-activated (IK) channel proteins of the present inventioncomprise at least N amino acids from SEQ ID NO:32 and conservativelymodified variants thereof, where N is any one of the integers selectedfrom the group consisting of from 10 to 600 and the sequence is uniqueto the protein of origin.

[0099] Typically, the calcium-activated potassium channel proteins andspecific peptides are at least 15, 25, 35, or 50-amino acids in length,more preferably at least 100, 200, 300, 400, or 500 amino acids inlength, and most preferably the full length of SEQ ID NOS: 1, 2, 3, 4,19, 20, 32, 43, or 47, or conservatively modified variants thereof.Thus, the present invention provides full-length and subsequences of SEQID NO:1, 2, 3, 4, 19, 20, 32, 43, and 47 and full-length andsubsequences of conservatively modified variants of SEQ ID NO:1, 2, 3,4, 19, 20, 32, 43, and 47. A “full-length” sequence of SEQ ID NO:1, 2,3, 4, 19, 20, 32, 43, or 47 means the sequence of SEQ ID NO:1, 2, 3, 4,19, 20, 32, 43 or 47, respectively. A “full-length” sequence of aconservatively modified variant of SEQ ID NO:1, 2, 3, 4, 19, 20, 32, 43or 47 means a conservatively modified variant of SEQ ID NO:1, 2, 3, 4,19, 20, 32, 43 or 47 respectively. The calcium-activated potassiumchannel proteins and peptides of the present invention can be used asimmunogens for the preparation of immunodiagnostic probes for assessingincreased or decreased expression of calcium-activated potassiumchannels in drug screening assays.

[0100] The calcium-activated potassium channel proteins of the presentinvention also include proteins which have substantial identity (i.e.,similarity) to a calcium-activated potassium channel protein of at leastN amino acids from any one of the sequences selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47 andconservatively modified variants thereof, where N is any one of theintegers selected from the group consisting of 10 to 600. Generally, thecalcium-activated potassium channel proteins are at least 50, typicallyat least 100, preferably at least 200, more preferably at least 300, andmost preferably at least 400 amino acid residues in length. Typically,the substantially similar or conservatively modified variant of thecalcium-activated potassium SK or IK channel protein is a eukaryoticprotein, preferably from a multicellular eukaryotes such as insects,protozoans, birds, fishes, amphibians, reptiles, or mammals.

[0101] The SK channel proteins which are substantially identical to, ora conservatively modified variant of, an SK channel protein having asequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:43 and SEQ ID NO:47 willspecifically react, under immunologically reactive conditions, with animmunoglobulin reactive to an SK channel protein selected from the groupconsisting of SEQ ID NO:I, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:43, and SEQ ID NO:47.

[0102] Similarly, IK channel proteins which are substantially identicalto, or a conservatively modified variant of, an IK channel proteinhaving a sequence selected from SEQ ID NO.32 will specifically react,under immunologically reactive conditions, with an immunoglobulinreactive to an IK channel protein such as SEQ ID NO:32. A variety ofimmunoassay formats may be used to assess such an immunologicallyspecific reaction including, for example, ELISA, competitiveimmunoassays, radioimmunoassays, Western blots, indirectimmunofluorescent assays and the like.

[0103] Alternatively, the SK channel proteins which are substantiallyidentical to, or are a conservatively modified variant of, an SK channelprotein having a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:43, and SEQ IDNO:47 will comprise an amino acid sequence which has any one of thevalues from 60% to 100% similarity to a comparison window within thecore sequence (or “core region”) of an SK channel protein selected fromthe group consisting of SEQ ID NOS:1, 2, 3, 4, 19, 20, 43, and 47. IKchannel proteins which are substantially identical to, or are aconservatively modified variant of, an IK channel protein having thesequence of SEQ ID NO:32 will comprise an amino acid sequence which hasany one of the values from 60% to 100% similarity to a comparison windowwithin the core sequence (or “core region”) of the IK channel proteinhIK1.

[0104] Thus, similarity is determined by reference to the core region orsubsequence thereof. The core region of hSK1 (SEQ ID NO:1) is from aminoacid residue 124 through 451 (SEQ ID NO:27). The core region of rSK2(SEQ ID NO:2) is from amino acid residue 135 through 462. The coreregion of truncated rSK3 (SEQ ID NO:3) is from amino acid residue 109through 436. The core region of N-terminal extended rSK3 (SEQ ID NO:43)is from 288-615. The core region of rSK1 (SEQ ID NO:4) is defined by theregion which aligns with the foregoing regions. The core region of hSK2(SEQ ID NO:19) is from amino acid residue 134 through 461. The coreregion of truncated hSK3 (SEQ ID NO:20) is from amino acid residue 109through 436. The core region of N-terminal extended hSK3 (SEQ ID NO:47)is from 238-465. Thus, the core region of SEQ ID NOS: 1-4, 19, 20, 43and 47 are inclusive of and defined by the amino acid residuesubsequences LSDYALIFGM (SEQ ID NO:17) at the amino proximal end andQRKFLQAIHQ (SEQ ID NO:18) at the carboxyl proximal end. The core regionof hIK1 (SEQ ID NO:32) is amino acids 25 through 351. A subsequence ofthe core region has a length of any one of the numbers from 10 to thelength of a core sequence of SEQ ID NOS: 1, 2, 3, 4, 19, 20, 32, 43 or47. Preferably, SK or IK channel proteins comprise an amino acidsequence having at least 90% similarity over a comparison window of 20contiguous amino acids from within the core sequence.

[0105] Similarity is also determined by reference to functionalcharacteristics of the calcium activated channel protein. For example,the present invention provides several SK3 amino acid sequences, whichwhen expressed have virtually identical currents. cDNAs encoding rSK3have been isolated in two different forms. The first, SEQ ID NO:44encoding SEQ ID NO:43, is the endogenous rSK3 or N-terminal extendedrSK3. The second, SEQ ID NO:16, encoding SEQ ID NO:3, is truncatedrelative to SEQ ID NO:43 at the N-terminus. Truncated rSK3 protein (SEQID NO:3) also has a different C-terminus, in which the last 9 aminoacids of SEQ ID NO:43 are replaced with 5 different amino acids.Although these sequences differ at both the N- and C-terminus, theyexpress virtually identical currents. Since the N-terminal extended andtruncated SK3 express the same current, the N-terminal extension notessential to channel function per se but is likely involved in targetingthe protein to a specific location in the cell.

[0106] Similarly, two cDNAs for hSK3 have been identified: N-terminalextended hSK3 (SEQ ID NO:48, encoding SEQ ID NO:47) and truncated hSK3(SEQ ID NO:22, encoding SEQ ID NO:20). In addition, a similar N-terminalextension may exist for SK2. Genomic sequences from the mouse for bothSK2 and SK3 demonstrate that both have an extended open reading frame,which is contiguous with the amino acids sequences for which functionalcurrent expression has been demonstrated. Thus, substantially identicalSK channel proteins, or conservatively modified variants thereof, arealso identified on the basis of functional characteristics.

[0107] The present invention provides functional SK and IK channelproteins and subsequences thereof. Functional SK channels of the presentinvention have a unitary conductance of between 2 and 60 pS, moreusually 5 and 25 pS, and molecular weights between 40 and 100 Kd foreach of the SK channel proteins which make up the SK channel, moreusually 50 to 80 kD. Functional IK channels have a unitary conductanceof between 20 and 80 pS, and often 30 to 60 pS. Unitary conductance maybe conveniently determined using inside-out or outside-out patch clampconfigurations. These configurations are particularly indicated for thestudy of the biophysics of ionic channels (kinetics, conductivity,selectivity, mechanism of permeation and block). Patch clamp methods arewell known in the art. See, e.g., the review of Franciolini, Patch clamptechnique and biophysical study of membrane channels, Experientia,42(6):589-594 (1986); and Sakmann et al., Patch clamp techniques forstudying ionic channels in excitable membranes, Annual Review ofPhysiology, 46:455-472 (1984).

[0108] The isolated SK and IK proteins within the scope of the presentinvention include those which when full-length and expressed in a cellfrom a quiet line, define a functionality and pharmacology indicative ofan SK channel or IK. channel, respectively. A quiet line is a cell linethat in its native state (e.g., not expressing recombinant SK or IKchannels) has low or uninteresting electric activity, e.g., a CHO cellline. For example, a control cell (without expression of a putative SKchannel of the present invention) and an experimental cell (expressing aputative SK channel) are maintained under conditions standard formeasurement of electrophysiological paramaters as provided in theworking examples disclosed herein. Each cell is treated with a calciumionophore. Exemplary ionophores include, but are not limited to, suchstandard compounds as ionomycin (Sigma Chemical Co.) or A23187 (SigmaChemical Co.). A cell is often treated with an ionophore at aconcentration of about 1 μM.

[0109] Subsequently, electrophysiological measurements of the cells aretaken to detect induction of a potassium current (e.g., by radiotracer),or a change in conductance of the cell (e.g., by patch clamp), or achange in voltage (e.g., by fluorescent dye). If the presence an ionchannel is indicated by a calcium induced change, subsequent tests areused to characterize the channel as an SK channel of the presentinvention. Preferably, at least two characteristics are determined, morepreferably at least 3, or 4 are determined. Characteristics of SKchannels of the present invention are disclosed more fully herein.

[0110] For example, a cell expressing an SK channel of the presentinvention can have a conductance of between 2 to 30 pS, often between 2to 25 pS, can, but not necessarily, exhibit block by apamin at a rangefrom 10 pM to about 100 nM, can comprise an SK channel protein of about40 to 80 kD, can exhibit sequence similarity of at least 60%, and morepreferably at least 70%, 80%, 90% or 95% in an alignment with the coreregions of the exemplary SK channel proteins disclosed herein, and canbe specifically reactive, under immunologically reactive conditions,with an antibody raised to an exemplary SK or IK channel disclosedherein (e.g., SEQ ID NO:1-4, 19, 20, 32, 43 and 47). Such standardmethods aid in the identification of SK proteins of the presentinvention. Cells expressing an IK channel have the same functionalcharacteristics except they are blocked by CTX but not blocked by IBX orapamin and have a unitary conductance of between 20 and 80, often 35 to40 pS.

[0111] Solid phase synthesis of SK or IK channel proteins of less thanabout 50 amino acids in length may be accomplished by attaching theC-terminal amino acid of the sequence to an insoluble support followedby sequential addition of the remaining amino acids in the sequence.Techniques for solid phase synthesis are described by Barany andMerrifield, Solid-Phase Peptide Synthesis; pp.3-284in The Peptides:Analysis, Synthesis, Biology. Vol. 2: Special Methods in PeptideSynthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156(1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed.Pierce Chem. Co., Rockford, Ill. (1984). SK or IK channel proteins ofgreater length may be synthesized by condensation of the amino andcarboxy termini of shorter fragments. Methods of forming peptide bondsby activation of a carboxy terminal end (e.g., by the use of thecoupling reagent N,N′-dicycylohexylcarbodiimide)) is known to those ofskill.

Obtaining Nucleic Acids Encoding Calcium-Activated Potassium ChannelProteins

[0112] The present invention provides isolated nucleic acids of RNA,DNA, or chimeras thereof, which encode calcium activated, SK channelproteins (“SK channel protein nucleic acids”) or calcium activated, IKchannel proteins (“IK channel protein nucleic acids) as discussed morefully above. Nucleic acids of the present invention can be used asprobes, for example, in detecting deficiencies in the level of mRNA,mutations in the gene (e.g., substitutions, deletions, or additions),for monitoring upregulation of SK or IK channels in drug screeningassays, or for recombinant expression of SK or IK channel proteins foruse as immunogens in the preparation of antibodies.

[0113] Nucleic acids encoding the calcium-activated potassium channelproteins of the present invention can be made using standard recombinantor synthetic techniques. With the amino acid sequences of the SK or IKchannel proteins herein provided, one of skill can readily construct avariety of clones containing functionally equivalent nucleic acids, suchas nucleic acids which encode the same protein. Cloning methodologies toaccomplish these ends, and sequencing methods to verify the sequence ofnucleic acids are well known in the art. Examples of appropriate cloningand sequencing techniques, and instructions sufficient to direct personsof skill through many cloning exercises are found in Sambrook, et al.,Molecular Cloning: A Laboratory Manual (2nd Ed., Vols. 1-3, Cold SpringHarbor Laboratory (1989)), Methods in Enzymology, Vol. 152: Guide toMolecular Cloning Techniques (Berger and Kimmel (eds.), San Diego:Academic Press, Inc. (1987)), or Current Protocols in Molecular Biology,(Ausubel, et al. (eds.), Greene Publishing and Wiley-Interscience, NewYork (1987). Product information from manufacturers of biologicalreagents and experimental equipment also provide information useful inknown biological methods. Such manufacturers include the SIGMA chemicalcompany (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.), PharmaciaLKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (PaloAlto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.(Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and AppliedBiosystems (Foster City, Calif.), as well as many other commercialsources known to one of skill.

[0114] 1. Isolation of SK and IK Channel Proteins by Nucleic AcidHybridization

[0115] The isolated nucleic acid compositions of this invention, whetherRNA, cDNA, genomic DNA, or a hybrid of the various combinations, areisolated from biological sources or synthesized in vitro.Deoxynucleotides can be prepared by any suitable method including, forexample, cloning and restriction of appropriate sequences or directchemical synthesis by methods such as the phosphotriester method ofNarang et al. Meth. Enzymol. 68: 90-99 (1979); the phosphodiester methodof Brown et al., Meth. Enzymol. 68: 109-151 (1979); thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859-1862 (1981); the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers (1981), Tetrahedron Letts.,22(20):1859-1862, e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter et al. (1984) Nucleic Acids Res.,12:6159-6168; and, the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

[0116] Nucleic acids encoding an SK channel protein of SEQ ID NO:1 maybe obtained by amplification of a human hippocampal cDNA library usingisolated nucleic acid primers having the sequence:ATGCCGGGTCCCCGGGCGGCCTGC (SEQ ID NO:5) and TCACCCGCAGTCCGAGGGGGCCAC (SEQID NO:6). Nucleic acids encoding an SK channel protein of SEQ ID NO:2may be obtained by amplification of a rat brain cDNA library usingisolated nucleic acid primers having the sequence:

[0117] ATGAGCAGCTGCAGGTACAACGGG (SEQ ID NO:7) andCTAGCTACTCTCAGATGAAGTTGG (SEQ ID NO:8). Nucleic acids encoding an SKchannel protein of SEQ ID NO:43 may be obtained by amplification of arat brain cDNA library using isolated nucleic acid primers having thesequence:

[0118] ATGAGCTCCTGCAAATACAGCGGT (SEQ ID NO:9) and TTAGCAACTGCTTGAACTTG(SEQ ID NO:10). Nucleic acids encoding an SK channel protein of SEQ IDNO:4 may be obtained by amplification of a rat brain cDNA library usingisolated nucleic acid primers having the sequenceTCAGGGAAGCCCCCGACCGTCAGT (SEQ ID NO:11) and TCACCCACAGTCTGATGCCGTGGT(SEQ ID NO:12). Nucleic acids encoding an SK channel protein of SEQ IDNO:19 may be obtained by amplification of a human hippocampal cDNAlibrary using isolated nucleic acid primers having the sequence:

[0119] ATGAGCAGCTGCAGGTACAACG (SEQ ID NO:23) and CTAGCTACTCTCTGATGAAGTTG(SEQ ID NO:24). Nucleic acids encoding an SK channel protein of SEQ IDNO:20 (hSK3) may be obtained by amplification of a human hippocampalcDNA library using isolated nucleic acid primers having the sequence:

[0120] ATGAGCTCCTGCAAGTATAGC (SEQ ID NO:25) and TTAGCAACTGCTTGAACTTGTG(SEQ ID NO:26). Nucleic acids encoding the IK channel protein of SEQ IDNO:32 may be obtained by amplification of a human pancreas cDNA libraryusing isolated nucleic acid primer pairs having the sequence: (SEQ IDNOS:38 and 39) and (SEQ ID NOS:40 and 41).

[0121] The isolated nucleic acids of the present invention may becloned, or amplified by in vitro methods, such as the polymerase chainreaction (PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-sustained sequence replicationsystem (SSR). A wide variety of cloning and in vitro amplificationmethodologies are well-known to persons of skill. Examples of thesetechniques and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al. (1989) MolecularCloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor Press, NY, (Sambrook et al.); CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashion etal., U.S. Pat. No. 5,017,478; and Carr, European Patent No.0,246,864.

[0122] Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No.4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & LevinsonOct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173; Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.Clin. Chem., 35:1826; Landegren et al., (1988) Science, 241:1077-1080;Van Brunt (1990) Biotechnology, 8: 291-294; Wu and Wallace, (1989) Gene,4: 560; and Barringeretal. (1990) Gene, 89:117.

[0123] Isolated nucleic acids encoding SK channel proteins comprise anucleic acid sequence encoding an SK channel protein selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:19, SEQ ID NO:20, and subsequences thereof. In preferredembodiments, the isolated nucleic acid encoding an SK channel protein isselected from the group consisting of: SEQ ID NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:22, and subsequencesthereof.

[0124] Isolated nucleic acids encoding IK channel proteins comprise anucleic acid sequence encoding an IK channel protein such as SEQ IDNO:32, and subsequences thereof. In preferred embodiments, the isolatednucleic acid encoding an IK channel protein is SEQ ID NO:31 andsubsequences thereof.

[0125] In addition to the isolated nucleic acids identified herein, theinvention also includes other isolated nucleic acids encodingcalcium-activated potassium channel proteins which selectivelyhybridize, under stringent conditions, to a nucleic acid encoding aprotein selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQID NO:43 and SEQ ID NO:47, and subsequences thereof. Generally, theisolated nucleic acid encoding a calcium-activated potassium channelprotein of the present invention will hybridize under at least moderatestringency hybridization conditions to a nucleic acid sequence from SEQID NOS: 13, 14, 15, 16, 21, 22, 31, 44, or 48 which encodes the coreregion or subsequence thereof. Alternatively, or additionally, theisolated nucleic acid encoding the calcium-activated potassium channelprotein will encode an amino acid sequence of at least 60%, 70%, 80%, or90% similarity over the length of the core region. Conveniently, thenucleic acid encoding a subsequence of the core region is obtained fromSEQ ID NOS: 13, 14, 15, 16, 21, 22, 32, 44, or 48 and is at least anyone of from 15 to 400 nucleotides in length, and generally at least 250or 300 nucleotides in length; preferably the nucleic acid will encodethe entire core sequence. The nucleic acid sequence, or subsequencethereof, encoding the calcium-activated potassium channel proteincomprises at least N′ nucleotides in length, where N′ is any one of theintegers selected from the group consisting of from 18 to 2000. Thus,the nucleic acids of the present invention comprise genomic DNA andnuclear transcripts encoding SK and IK channel proteins.

[0126] Where the nucleic acid encoding an SK or IK channel protein is tobe used as nucleic acid probes, it is often desirable to label thenucleic acid with detectable labels. The labels may be incorporated byany of a number of means well known to those of skill in the art.However, in a preferred embodiment, the label is simultaneouslyincorporated during the amplification step in the preparation of thenucleic acids. Thus, for example, polymerase chain reaction (PCR) withlabeled primers or labeled nucleotides will provide a labeledamplification product. In another preferred embodiment, transcriptionamplification using a labeled nucleotide (e.g., fluorescein-labeled UTPand/or CTP) incorporates a label into the transcribed nucleic acids.

[0127] Alternatively, a label maybe added directly to an originalnucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to theamplification product after the amplification is completed. Means ofattaching labels to nucleic acids are well known to those of skill inthe art and include, for example nick translation or end-labeling (e.g.,with a labeled RNA) by phosphorylation of the nucleic acid andsubsequent attachment (ligation) of a nucleic acid linker joining thesample nucleic acid to a label (e.g., a fluorophore).

[0128] Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, radioisotopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include biotinfor staining with labeled streptavidin conjugate, magnetic beads,fluorescent dyes (e.g., fluorescein, texas red, rhodamine, greenfluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S,¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and colorimetriclabels such as colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, latex, etc.) beads. Patents teaching the useof such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0129] Means of detecting such labels are well known to those of skillin the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted light. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

[0130] The probes are used to screen genomic or cDNA libraries from anysource of interest including specific tissues (e.g., heart, brain,pancreas) and animal source such as rat, human, bird, etc. Screeningtechniques are known in the art and are described in the general textscited above such as in Sambrook and Ausubel.

[0131] 2. Isolation of SK and IK Channel Proteins by Immunoscreening

[0132] In addition to using nucleic acid probes for identifying novelforms of the protein claimed herein, it is possible to use antibodies toprobe expression-libraries. This is a well known technology. (See Youngand Davis, 1982 Efficient isolation of genes using antibody probes Proc.Natl. Acad. Sci., U.S.A. 80:1194-1198.) In general, a cDNA expressionlibrary maybe prepared from commercially available kits or using readilyavailable components. Phage vectors are preferred, but a variety ofother vectors are available for the expression of protein. such vectorsinclude but are not limited to yeast, animal cells and Xenopus oocytes.One selects mRNA from a source that is enriched with the target proteinand creates cDNA which is then ligated into a vector and transformedinto the library host cells for immunoscreening. Screening involvesbinding and visualization of antibodies bound to specific proteins oncells or immobilized on a solid support such as nitrocellulose or nylonmembranes. Positive clones are selected for purification to homogeneityand the isolated cDNA then prepared for expression in the desired hostcells. A good general review of this technology can be found in Methodsof Cell Biology Vol 37 entitled Antibodies in Cell Biology, Ed. D J Asaipp 369-382, 1993.

[0133] When choosing to obtain calcium activated channel proteinsantibodies selective for the entire protein or portions can be used.Suitable peptide sequences include, but are not limited to,GHRRALFEKRKRLSDY (SEQ ID NO:28), FTDASSRSIGAL (SEQ ID NO:29), andARKLELTKAEKHVHNFMMDTQLTKR (SEQ ID NO:30) or ARKLELTKAEKHVHNFMMDTQLTK(SEQ ID NO:42).

Nucleic Acid Assays

[0134] This invention also provides methods of detecting and/orquantifying SK or IK channel protein expression by assaying for the genetranscript (e.g., nuclear RNA, mRNA). The assay can be for the presenceor absence of the normal gene or gene product, for the presence orabsence of an abnormal gene or gene product, or quantification of thetranscription levels of normal or abnormal SK or IK channel protein geneproduct.

[0135] In a preferred embodiment, nucleic acid assays are performed witha sample of nucleic acid isolated from the organism to be tested. In thesimplest embodiment, such a nucleic acid sample is the total mRNAisolated from a biological sample. The nucleic acid (e.g., eithergenomic DNA or mRNA) may be isolated from the sample according to any ofa number of methods well known to those of skill in the art.

[0136] Methods of isolating total DNA or mRNA for use in, inter alia, anucleic acid assay are well known to those of skill in the art. Forexample, methods of isolation and purification of nucleic acids aredescribed in detail in Chapter 3 of Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed.Elsevier, N.Y. (1993). One of skill will appreciate that wherealterations in the copy number of the gene encoding an SK or IK channelprotein is to be detected genomic DNA is preferably isolated.Conversely, where expression levels of a gene or genes are to bedetected, preferably RNA (mRNA) is isolated.

[0137] Frequently, it is desirable to amplify the nucleic acid sampleprior to hybridization. One of skill in the art will appreciate thatwhatever amplification method is used, if a quantitative result isdesired, care must be taken to use a method that maintains or controlsfor the relative frequencies of the amplified nucleic acids. Methods of“quantitative” amplification are well known to those of skill in theart. For example, quantitative PCR involves simultaneously co-amplifyinga known quantity of a control sequence using the same primers. Thisprovides an internal standard that may be used to calibrate the PCRreaction. The high density array may then include probes specific to theinternal standard for quantification of the amplified nucleic acid.Detailed protocols for quantitative PCR are provided in PCR Protocols, AGuide to Methods and Applications, Innis et al., Academic Press, Inc.N.Y., (1990).

[0138] The method of detecting the presence of a nucleic acid sequenceencoding an SK channel protein generally comprises: (a) contacting thebiological sample, under stringent hybridization conditions, with anucleic acid probe comprising a nucleic acid segment which selectivelyhybridizes to a nucleic acid sequence (target) encoding an SK channelprotein selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:43, andSEQ ID NO:47; (b) allowing the probe to specifically hybridize to thenucleic acid encoding an SK channel protein to form a hybridizationcomplex, wherein detection of the hybridization complex is an indicationof the presence of the SK nucleic acid sequence in the sample. Detectionof an IK channel protein is accomplished in a similar fashion using anucleic acid segment which selectively hybridizes to a nucleic acidsequence encoding an IK channel protein of SEQ ID NO:32.

[0139] The nucleic acid segment of the probe is a subsequence of atleast. N″ contiguous nucleotides in length from a nucleic acid encodingan SK channel selected from the group consisting of SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:44, and SEQ ID NO:48, and complementary sequences thereof. N″ is anany one of the integers selected from the group consisting of each ofthe integers from 15 to 1500. For detecting the presence of an IKchannel protein the nucleic acid segment is a subsequence of at least N″contiguous nucleotides in length from a nucleic acid encoding an IKchannel of SEQ ID NO:31. “Contiguous nucleotides” from a referencednucleic acid-means a sequence of nucleotides-having-the-same order anddirectly adjacent to the same nucleotides (i.e., without additions ordeletions) as in the referenced nucleic acid. Typically, the nucleicacid segment is at least 18 nucleotides in length. The preferred lengthof the nucleic acid probe is from 24 to 200 nucleotides in length.

[0140] In particularly preferred embodiments, the nucleic acid segmentis derived from a nucleic acid which encodes a core region from aprotein selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 19,20, 32, 43 and 47. Conveniently, the nucleic acid which encodes the coreregion is a subsequence of a nucleic acid selected from the groupconsisting of: SEQ ID NOS: 13, 14, 15, 16, 21, 22, 31, 44, 48, andcomplementary sequences thereof. Usually, and particularly forcross-species hybridization, the nucleic acid segment would encode anamino acid sequence from within the core region and will be at least 250nucleotides in length, most preferably will encode the entirety of thecore region, and/or will hybridize to the target sequence under moderatestringency hybridization conditions.

[0141] Those of skill will appreciate that nucleic acid sequences of theprobe will be chosen so as not to interfere in the selectivehybridization of the nucleic acid segment to the target. Thus, forexample, any additional nucleotides attached to the nucleic acid segmentwill generally be chosen so as not to selectively hybridize, understringent conditions, to the nucleic acid target (potential falsenegative), nor to nucleic acids not encoding an SK or IK channelproteins or peptides (potential false positive). The use of negative andpositive controls to ensure selectivity and specificity is known tothose of skill. In general, the length of the probe should be kept tothe minimum length necessary to achieve the desired results. The lengthof the nucleic acid encoding an SK or IK channel protein or peptide(i.e., the “SK channel protein nucleic acid” or “IK channel proteinnucleic acid”, respectively) is discussed more fully, supra, but ispreferably at least 30 nucleotides in length.

[0142] A variety of nucleic acid hybridization formats are known tothose skilled in the art. For example, common formats include sandwichassays and competition or displacement assays. Hybridization techniquesare generally described in Berger and Kimmel, (1987), supra.; “NucleicAcid Hybridization, A Practical Approach” (Hames, B. D. and Higgins, S.J. (eds.), IRL Press, 1985; Gall and Pardue, (Proc. Natl. Acad. Sci.,U.S.A. 63:378-383 (1969)); and John, Burnsteil and Jones (Nature,223:582-587 (1969)).

[0143] Sandwich assays are commercially useful hybridization assays fordetecting or isolating nucleic acid sequences. Such assays utilize a“capture” nucleic acid covalently immobilized to a solid support and alabelled “signal” nucleic acid in solution. The biological sample willprovide the target nucleic acid. The “capture” nucleic acid probe andthe “signal” nucleic-acid probe hybridize with the target nucleic acidto form a sandwich“hybridization complex. To be effective, the signalnucleic acid cannot hybridize with the capture nucleic acid.

[0144] In in situ hybridization, the target nucleic acid is liberatedfrom its cellular surroundings in such as to be available forhybridization within the cell while preserving the cellular morphologyfor subsequent interpretation and analysis. The following articlesprovide an overview of the art of in situ hybridization: Singer et al.,Biotechniques 4(3):230-250 (1986); Haase et al., Methods in Virology,Vol. VII, pp. 189-226 (1984); Wilkinson, “The theory and practice of insitu hybridization ” In: In situ Hybridization, Ed. D. G. Wilkinson. IRLPress, Oxford University Press, Oxford, and Nucleic Acid Hybridization:A Practical Approach, Ed. Hames, B. D. and Higgins, S. J., IRL Press(1987).

[0145] Typically, labelled signal nucleic acids are used to detecthybridization. Complementary nucleic acids or signal nucleic acids maybe labelled by any one of several methods typically used to detect thepresence of hybridized oligonucleotides. The most common method ofdetection is the use of autoradiography with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P-labelled probes or the like. Other labels include ligands which bindto labelled antibodies, fluorophores, chemiluminescent agents, enzymes,and antibodies which can serve as specific binding pair members for alabelled ligand.

[0146] The label may also allow for the indirect detection of thehybridization complex. For example, where the label is a hapten orantigen, the sample can be detected by using antibodies. In thesesystems, a signal is generated by attaching fluorescent or enzymemolecules to the antibodies or, in some cases, by attachment to aradioactive label. (Tijssen, “Practice and Theory of EnzymeImmunoassays,” Laboratory Techniques in Biochemistry and MolecularBiology”(Burdon, van Knippenberg (eds.), Elsevier, pp. 9-20 (1985)).

[0147] The detectable label used in nucleic acids of the presentinvention may be incorporated by any of a number of means known to thoseof skill in the art, e.g., as discussed supra. Means of detecting suchlabels are well known to those of skill in the art.

[0148] The sensitivity of the hybridization assays may be enhancedthrough the use of a nucleic acid amplification system which multipliesthe target nucleic acid being detected. Examples of such systems includethe polymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods known in the art are the nucleic acidsequence based amplification (NASBA, Cangene, Mississauga, Ontario) andQ-Beta Replicase systems.

[0149] Those of skill will appreciate that abnormal expression levels orabnormal expression products (e.g., mutated transcripts, truncated ornon-sense proteins) are identified by comparison to normal expressionlevels and normal expression products. Normal levels of expression ornormal expression products can be determined for any particularpopulation, subpopulation, or group of organisms according to standardmethods known to those of skill in the art. Typically this involvesidentifying healthy organisms (i.e., organisms with a functional SK orIK channel protein as indicated by such properties as conductance andcalcium sensitivity) and measuring expression levels of the SK or IKchannel protein gene (as described herein) or sequencing the gene, mRNA,or reverse transcribed cDNA, to obtain typical (normal) sequencevariations. Application of standard statistical methods used inmolecular genetics permits determination of baseline levels ofexpression, and normal gene products as well as significant deviationsfrom such baseline levels.

Nucleic Acid Assay Kits

[0150] The nucleic acids of this invention can be included in a kitwhich can be used to determine in a biological sample the presence orabsence of the normal gene or gene product encoding an SK or IK channelof the present invention, for the presence or absence of an abnormalgene or gene product encoding an SK or IK channel, or quantification ofthe transcription levels of normal or abnormal SK or IK channel proteingene product. The kit typically includes a stable preparation of nucleicacid probes for performing the assay of the present invention. Further,the kit may also include a hybridization solution in either dry orliquid form for the hybridization of probes to target calcium-activatedpotassium channel proteins or calcium-activated potassium channelprotein nucleic acids of the present invention, a solution for washingand removing undesirable and non-hybridized nucleic acids, a substratefor detecting the hybridization complex, and/or instructions forperforming and interpreting the assay.

Expression of Nucleic Acids

[0151] Once the nucleic acids encoding an SK or IK channel protein ofthe present invention are isolated and cloned, one may express thedesired protein in a recombinantly engineered cell such as bacteria,yeast, insect (especially employing baculoviral vectors), and mammaliancells. A “recombinant protein” is a protein produced using cells that donot have in their native form an endogenous copy of the DNA able toexpress the protein. The cells produce the recombinant protein becausethey have beer genetically altered by the introduction of theappropriate isolated nucleic acid sequence (e.g., a vector comprising anSK or IK channel protein nucleic acid).

[0152] It is expected that those of skill in the art are knowledgeablein the numerous expression systems available for expression of DNAencoding SK or IK channel proteins. No attempt to describe in detail thevarious methods known for the expression of proteins in prokaryotes oreukaryotes will be made.

[0153] In brief summary, the expression of natural or synthetic nucleicacids encoding calcium-activated potassium channel proteins of thepresent invention will typically be achieved by operably linking the DNAor cDNA to a promoter (which is either constitutive or inducible),followed by incorporation into an expression vector. The vectors can besuitable for replication and integration in either prokaryotes oreukaryotes. Typical expression vectors contain transcription andtranslation terminators, initiation sequences, and promoters useful forregulation of the expression of the DNA encoding the SK or IK channelprotein. To obtain high level expression of a cloned gene, it isdesirable to construct expression vectors which contain, at the minimum,a strong promoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.One of skill would recognize that modifications can be made to an SK orIK channel protein without diminishing its biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

[0154] 1. Expression in Prokaryotes

[0155] Examples of regulatory regions suitable for this purpose in E.coli are the promoter and operator region of the E. coli tryptophanbiosynthetic pathway as described by Yanofsky, Bacteriol. 158:1018-1024(1984), and the leftward promoter of phage lambda (P_(L)) as describedby Herskowitz and Hagen, Ann. Rev. Genet., 14:399-445 (1980). Theinclusion of selection markers in DNA vectors transfected in E. coli isalso useful. Examples of such markers include genes specifyingresistance to ampicillin, tetracycline, or chloramphenicol. See,Sambrook, et al. for details concerning selection markers for use in E.coli.

[0156] The vector is selected to allow introduction into the appropriatehost cell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing SK channel proteins are availableusing E. coli, Bacillus sp. and Salmonella (Palva, et al., Gene22:229-235 (1983); Mosbach, et al., Nature 302:543-545 (1983)).

[0157] When expressing SK or IK channel proteins in S. typhimurium, oneshould be aware of the inherent instability of plasmid vectors. Tocircumvent this, the foreign gene can be incorporated into anonessential region of the host chromosome. This is achieved by firstinserting the gene into a plasmid such that it is flanked by regions ofDNA homologous to the insertion site in the Salmonella chromosome. Afterintroduction of the plasmid into the S. typhimurium, the foreign gene isincorporated into the chromosome by homologous recombination between theflanking sequences and chromosomal DNA.

[0158] An example of how this can be achieved is based on the his operonof Salmonella. Two steps are involved in this process. First, a segmentof the his operon must be deleted in the Salmonella strain selected asthe carrier. Second, a plasmid carrying the deleted his regiondownstream of the gene encoding the SK or IK channel protein istransfected into the his Salmonella strain. Integration of both the hissequences and a gene encoding an SK or IK channel protein occurs,resulting in recombinant strains which can be selected as his⁺.

[0159] Detection of the expressed protein is achieved by methods knownin the art and include, for example, radioimmunoassays, Western blottingtechniques or immunoprecipitation. Purification from E. coli can beachieved following procedures described in U.S. Pat. No. 4,511,503.

[0160] 2. Expression in Eukaryotes

[0161] A variety of eukaryotic expression systems such as yeast, insectcell lines, bird, fish, frog, and mammalian cells, are known to those ofskill in the art. As explained briefly below, SK or IK channel proteinsof the present invention may be expressed in these eukaryotic systems.Expression of SK or IK channels in eukaryotes is particularly preferred.

[0162] Synthesis of heterologous proteins in yeast is well known.Methods in Yeast Genetics, Sherman, F., et al., Cold Spring HarborLaboratory, (1982) is a well recognized work describing the variousmethods available to produce the protein in yeast. Suitable vectorsusually have expression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired. Forinstance, suitable vectors are described in the literature (Botstein, etal., 1979, Gene, 8:17-24; Broach, et al., 1979, Gene, 8:121-133).

[0163] Two procedures are used in transfecting yeast cells. In one case,yeast cells are first converted into protoplasts using zymolyase,lyticase or glusulase, followed by addition of DNA and polyethyleneglycol (PEG). The PEG-treated protoplasts are then regenerated in a 3%agar medium under selective-conditions. Details-of this procedure aregiven in the papers by J. D. Beggs, 1978, Nature (London), 275:104-109;and Hinnen, A., et al., 1978, Proc. Natl. Acad. Sci. USA, 75:1929-1933.The second procedure does not involve removal of the cell wall. Insteadthe cells are treated with lithium chloride or acetate and PEG and puton selective plates (Ito, H., et al., 1983, J. Bact., 153:163-168).

[0164] The calcium-activated potassium channel proteins of the presentinvention, once expressed, can be isolated from yeast by lysing thecells and applying standard protein isolation techniques to the lysates.The monitoring of the purification process can be accomplished by usingWestern blot techniques or radioimmunoassay of other standardimmunoassay techniques.

[0165] The sequences encoding the calcium-activated potassium channelproteins can also be ligated to various expression vectors for use intransfecting cell cultures of, for instance, mammalian, insect, bird,amphibian, or fish origin. Illustrative of cell cultures useful for theproduction of the peptides are mammalian cells. Mammalian cell systemsoften will be in the form of monolayers of cells although mammalian cellsuspensions may also be used. A number of suitable host cell linescapable of expressing intact proteins have been developed in the art,and include the HEK293, BHK21, and CHO cell lines, and various humancells such as COS cell lines, HeLa cells, myeloma cell lines, Jurkatcells. In some embodiments, Xenopus oocytes are used. Those of skillwill recognize that preferred cell lines for expressing SK or IKchannels substantially lack conductances which compete with thoseprovided by the calcium-activated potassium channels of the presentinvention (i.e., “quiet lines”). Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen et al. (1986)Immunol. Rev. 89:49), and necessary processing information sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites(e.g., an SV40 large T Ag poly A addition site), and transcriptionalterminator sequences. Other animal cells useful for production of SKchannel proteins are available, for instance, from the American TypeCulture Collection Catalogue of Cell Lines and Hybridomas (7th edition,1992).

[0166] Appropriate vectors for expressing SK or IK channel proteins ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See Schneider J.Embryol. Exp. Morphol. 27:353-365 (1987).

[0167] As indicated above, the vector, e.g., a plasmid, which is used totransfect the host cell, preferably contains DNA sequences to initiatetranscription and sequence to control the translation of the protein.These sequences are referred to as expression control sequences.

[0168] As with yeast, when higher animal host cells are employed,polyadenlyation or transcription terminator sequences from knownmammalian genes need to be incorporated into the vector. An example of aterminator sequence is the polyadenlyation sequence from the bovinegrowth hormone gene. Sequences for accurate splicing of the transcriptmay also be included. An example of a splicing sequence is the VP1intron from SV40 (Sprague, J. et al., 1983, J. Virol. 45: 773-781).

[0169] Additionally, gene sequences to control replication in the hostcell may be incorporated into the vector such as those found in bovinepapilloma virus type-vectors. Saveria-Campo, M., 1985, “Bovine Papillomavirus DNA a Eukaryotic Cloning Vector” in DNA Cloning Vol. II aPractical Approach Ed. D. M. Glover, IRL Press, Arlington, Va. pp.213-238.

[0170] The host cells are competent or rendered competent fortransfection by various means. There are several well-known methods ofintroducing DNA into animal cells. These include: calcium phosphateprecipitation, fusion of the recipient cells with bacterial protoplastscontaining the DNA, treatment of the recipient cells with liposomescontaining the DNA, DEAE dextran, electroporation and micro-injection ofthe DNA directly into the cells. The transfected cells are cultured bymeans well known in the art. Biochemical Methods in Cell Culture andVirology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc., (1977). Theexpressed proteins are recovered by well known mechanical, chemical orenzymatic means.

Purification of Expressed Peptides

[0171] The SK or IK channel proteins of the present invention which areproduced by recombinant DNA technology may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced SK or IK channel proteins can be directly expressed orexpressed as a fusion protein. The recombinant calcium-activatedpotassium channel protein of the present invention is purified by acombination of cell lysis (e.g., sonication) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant calcium-activated potassium channel protein.

[0172] The calcium-activated potassium channel proteins of thisinvention, recombinant or synthetic, may be purified to substantialpurity by standard techniques well known in the art, including selectiveprecipitation with such substances as ammonium sulfate, columnchromatography, immunopurification methods, and others. See, forinstance, R. Scopes, Protein Purification: Principles and Practice,Springer-Verlag: New York (1982); Deutscher, Guide to ProteinPurification, Academic Press, 1990. For example, the proteins of thisinvention may be purified by immunoaffinity columns using antibodiesraised to the SK or IK channel proteins as described herein.

Antibodies to Calcium-Activated Potassium Channel Proteins

[0173] Antibodies are raised to the SK or IK channel protein of thepresent invention, including individual, allelic, strain, or speciesvariants, and fragments thereof, both in their naturally occurring(full-length) forms and in recombinant forms. Additionally, antibodiesare raised to these proteins in either their native configurations or innon-native configurations. Anti-idiotypic antibodies can also begenerated. Many methods of making antibodies are known to persons ofskill. The following discussion is presented as a general overview ofthe techniques available; however, one of skill will recognize that manyvariations upon the following methods are known.

[0174] A. Antibody Production

[0175] A number of immunogens are used to produce antibodiesspecifically reactive with an SK or IK channel protein. An isolatedrecombinant, synthetic, or native SK or IK channel protein of 5 aminoacids in length or greater, and selected from a subsequence of SEQ IDNO:1, 2, 3, 4, 19, 20, 32, 43, or 47 are the preferred immunogens(antigen) for the production of monoclonal or polyclonal antibodies.Those of skill will readily understand that the calcium-activatedpotassium channel proteins of the present invention are typicallydenatured prior to formation of antibodies for screening expressionlibraries or other assays in which a putative calcium-activatedpotassium channel protein of the present invention is expressed ordenatured in a non-native secondary, tertiary, or quartenary structure.Exemplary proteins for use as immunogens include, but are not limitedto, GHRRALFEKRKRLSDY (SEQ ID NO:28), FTDASSRSIGAL (SEQ ID NO:29),ARKLELTKAEKHVHNFMMDTQLTKR (SEQ ID NO:30), and ARKLELTKAEKHVHNFMMDTQLTK(SEQ ID NO:42). In one class of preferred embodiments, an immunogenicprotein conjugate is also included as an immunogen. Naturally occurringSK or IK channel proteins are also used either in pure or impure form.

[0176] The SK or IK channel protein is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies can be generated for subsequent use in immunoassays tomeasure the presence and quantity of the calcium-activated potassiumchannel protein. Methods of producing polyclonal antibodies are known tothose of skill in the art. In brief, an immunogen (antigen), preferablya purified SK or IK channel protein,an SK or IK channel protein coupledto an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin,etc.), or an SK or IK channel protein incorporated into an immunizationvector such as a recombinant vaccinia virus (see, U.S. Pat. No.4,722,848) is mixed with an adjuvant and animals are immunized with themixture. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the calcium-activated potassium channel protein of interest. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theSK or IK channel protein is performed where desired (see, e.g., Coligan(1991) Current Protocols in Immunology Wiley/Greene, NY; and Harlow andLane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press,NY).

[0177] Antibodies, including binding fragments and single chainrecombinant versions thereof, against predetermined fragments of SK orIK channel protein are raised by immunizing animals, e.g., withconjugates of the fragments with carrier proteins as described above.Typically, the immunogen of interest is an SK or IK channel protein ofat least about 5 amino acids, more typically the SK or IK channelprotein is 10 amino acids in length, preferably, 15 amino acids inlength and more preferably the calcium-activated potassium channelprotein is 20 amino acids in length or greater. The peptides aretypically coupled to a carrier protein (e.g., as a fusion protein), orare recombinantly expressed in an immunization vector. Antigenicdeterminants on peptides to which antibodies bind are typically 3 to 10amino acids in length.

[0178] Monoclonal antibodies are prepared from cells secreting thedesired antibody. Monoclonals antibodies are screened for binding to anSK or IK channel protein from which the immunogen was derived. Specificmonoclonal and polyclonal antibodies will usually bind with a K_(D) ofat least about 0.1 mM, more usually at least about 50 μM, and mostpreferably at least about 1 μM or better.

[0179] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies are found in, e.g., Stites et al. (eds.) Basic andClinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane, Supra; Goding(1986) Monoclonal Antibodies. Principles and Practice (2d ed.) AcademicPress, New York, N.Y.; and Kohler and Milstein (1975) Nature 256:495-497. Summarized briefly, this method proceeds by injecting an animalwith an immunogen comprising an SK or IK channel protein. The animal isthen sacrificed and cells taken from its spleen, which are fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

[0180] Alternative methods of immortalization include transfection withEpstein Barr Virus, oncogenes, or retroviruses, or other methods knownin the art. Colonies arising from single immortalized cells are screenedfor production of antibodies of the desired specificity and affinity forthe antigen, and yield of the monoclonal antibodies produced by suchcells is enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate (preferably mammalian) host. The SK orIK channel proteins and antibodies of the present invention are usedwith or without modification, and include chimeric antibodies such ashumanized murine antibodies.

[0181] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phage or similar vectors (see, e.g., Huse etal. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341:544-546; and Vaughan et al. (1996) Nature Biotechnology, 14: 309-314).Alternatively, high avidity human monoclonal antibodies can be obtainedfrom transgenic mice comprising fragments of the unrearranged humanheavy and light chain Ig loci (i.e., minilocus transgenic mice).Fishwild et al., Nature Biotech., 14:845-851 (1996).

[0182] Frequently, the SK or IK channel proteins and antibodies will belabeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like. Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'lAcad. Sci. USA 86: 10029-10033.

[0183] The antibodies of this invention are also used for affinitychromatography in isolating SK or IK channel proteins. Columns areprepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysateis passed through the column, washed, and treated with increasingconcentrations of a mild denaturant, whereby purified SK or IK channelprotein are released.

[0184] The antibodies can be used to screen expression libraries forparticular expression products such as normal or abnormal human SK or IKchannel protein. Usually the antibodies in such a procedure are labeledwith a moiety allowing easy detection of presence of antigen by antibodybinding.

[0185] Antibodies raised against SK or IK channel protein can also beused to raise anti-idiotypic antibodies. These are useful for detectingor diagnosing various pathological conditions related to the presence ofthe respective antigens.

[0186] B. Human or Humanized (Chimeric) Antibody Production

[0187] The anti-SK or anti-IK channel protein antibodies of thisinvention can also be administered to a mammal (e.g., a human patient)for therapeutic purposes (e.g., as targeting molecules when conjugatedor fused to effector molecules such as labels, cytotoxins, enzymes,growth factors, drugs, etc.). Antibodies administered to an organismother than the species in which they are raised are often immunogenic.Thus, for example, murine antibodies administered to a human ofteninduce an immunologic response against the antibody (e.g., the humananti-mouse antibody (HAMA) response) on multiple administrations. Theimmunogenic properties of the antibody are reduced by altering portions,or all, of the antibody into characteristically human sequences therebyproducing chimeric or human antibodies, respectively.

[0188] i) Humanized (Chimeric) Antibodies

[0189] Humanized (chimeric) antibodies are immunoglobulin moleculescomprising a human and non-human portion. More specifically, the antigencombining region (or variable region) of a humanized chimeric antibodyis derived from a non-human source (e.g., murine) and the constantregion of the chimeric antibody (which confers biological effectorfunction to the immunoglobulin) is derived from a human source. Thehumanized chimeric antibody should have the antigen binding specificityof the non-human antibody molecule and the effector function conferredby the human antibody molecule. A large number of methods of generatingchimeric antibodies are well known to those of skill in the art (see,e.g., U.S. Pat. Nos: 5,502,167, 5,500,362, 5,491,088, 5,482,856,5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238,5,169,939, 5,081,235, 5,075,431, and 4,975,369). Detailed methods forpreparation of chimeric (humanized) antibodies can be found in U.S. Pat.No. 5,482,856.

[0190] ii) Human Antibodies

[0191] In another embodiment, this invention provides for fully humananti-SK channel protein antibodies. Human antibodies consist entirely ofcharacteristically human polypeptide sequences. The human anti-SK oranti-IK channel protein antibodies of this invention can be produced inusing a wide variety of methods (see, e.g., Larrick et al., U.S. Pat.No. 5,001,065, for review).

[0192] In preferred embodiments, the human anti-SK channel proteinantibodies of the present invention are usually produced initially intrioma cells. Genes encoding the antibodies are then cloned andexpressed in other cells, particularly, nonhuman mammalian cells. Thegeneral approach for producing human antibodies by trioma technology hasbeen described by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg,U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666.The antibody-producing cell lines obtained by this method are calledtriomas because they are descended from three cells; two human and onemouse. Triomas have been found to produce antibody more stably thanordinary hybridomas made from human cells.

[0193] The genes encoding the heavy and light chains of immunoglobulinssecreted by trioma cell lines are cloned according to methods, includingthe polymerase chain reaction, known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor, N.Y., 1989; Berger & Kimmel, Methods in Enzymology, Vol. 152:Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego,Calif., 1987; Co et al. (1992) J. Immunol., 148: 1149). For example,genes encoding heavy and light chains are cloned from a trioma's genomicDNA or cDNA produced by reverse transcription of the trioma's RNA.Cloning is accomplished by conventional techniques including the use ofPCR primers that hybridize to the sequences flanking or overlapping thegenes, or segments of genes, to be cloned.

Calcium-Activated Potassium Channel Protein Immunoassays

[0194] Immunoassays for SK and IK channel proteins can be used for atleast two different purposes. They can be used to determine therelatedness of the protein by virtue of their being able to cross-reactimmunologically or for detection of the presence or absense of thechannel proteins.

[0195] When determining if an unknown protein is related to the channelproteins of this invention, a variety of assays can be used. For exampleand preferred is a competitive immunoassay to test for cross-reactivity.For example, the protein of SEQ ID NO:2 or 32 can be immobilized to asolid-support. Proteins or peptides are added to the assay which competewith the binding of the antisera to the immobilized antigen. The abilityof the above proteins to compete with the binding of the antisera to theimmobilized protein is compared to the protein thought to be related tothe test protein.

[0196] To assure that the antisera being tested is specific orselectively binding to a particular protein, it will be tested forcross-reactivity to other closely related proteins. This allows for theproduction of sera that will distinguish between small, intermediate andlarge conductance channels. The percent crossreactivity for the aboveproteins can be calculated, using standard calculations. Those antiserawith less than 10% crossreactivity with each of the proteins listedabove are selected and pooled. The cross-reacting antibodies areoptionally removed from the pooled antisera by immunoabsorption with theabove-listed proteins.

[0197] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein to the claimed or prototype immunogen protein. In order to makethis comparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of protein required is less than twice the amount of theprototype protein, then the second protein is said to specifically bindto an antibody generated to the prototype immunogen. Where theantibodies are generated to a short peptide, the test proteins areoptionally denatured to fully test for selective binding. In situationswhere the target peptide is not readily accessible to the antibodiesbecause the target peptide is part of a larger protein, it is proper tomeasure the relatedness of test proteins against prototype proteins ofsimilar size, e.g., one would test a full length monomer against aprototype, full length monomer even though the antisera was generatedagainst a peptide of the prototype monomer. This simplifies the readingof the test results and avoids having to take into accountconformational problems and molecular weight/molar concentrations in thedetermination of the data generated from the competitive immunoassays.

[0198] Means of detecting the SK or IK channel proteins of the presentinvention are not critical aspects of the present invention. In apreferred embodiment, the SK or IK channel proteins are detected and/orquantified using any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Methods in Cell Biology Volume 37: Antibodies in Cell Biology,Asai, ed. Academic Press, Inc. New York (1993); Basic and ClinicalImmunology 7th Edition, Stites & Terr;,eds. (1991). Immunologicalbinding assays (or immunoassays) typically utilize a “capture agent” tospecifically bind to and often immobilize the analyte (in this case acalcium-activated potassium channel protein). The capture agent is amoiety that specifically binds to the analyte. In a preferredembodiment, the capture agent is an antibody that specifically binds acalcium-activated potassium channel protein(s) of the present invention.The antibody (anti-SK or anti-IK channel protein antibody) may beproduced by any of a number of means known to those of skill in the artas described herein.

[0199] Immunoassays also often utilize a labeling agent to specificallybind to and label the binding complex formed by the capture agent andthe analyte. The labeling agent may itself be one of the moietiescomprising the antibody/analyte complex. Thus, the labeling agent may bea labeled SK or IK channel protein or a labeled anti-SK or anti-IKchannel protein antibody. Alternatively, the labeling agent may be athird moiety, such as another antibody, that specifically binds to theantibody/SK or antibody/IK channel protein complex.

[0200] In a preferred embodiment, the labeling agent is a second SK orIK channel protein antibody bearing a label. Alternatively, the secondSK or IK channel protein antibody may lack a label, but it may, in turn,be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second can bemodified with a detectable moiety, such as biotin, to which a thirdlabeled molecule can specifically bind, such as enzyme-labeledstreptavidin.

[0201] Other proteins capable of specifically binding immunoglobulinconstant regions, such as protein A or protein G may also be used as thelabel agent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, andAkerstrom, et al. (1985) J. Immunol., 135: 2589-2542).

[0202] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, analyte, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10C to 40°C.

[0203] While the details of the immunoassays of the present inventionmay vary with the particular format employed, the method of detecting anSK or IK channel protein in a biological sample generally comprises thesteps of contacting the biological sample with an antibody whichspecifically reacts, under immunologically reactive conditions, to theSK or IK channel protein. The antibody is allowed to bind to the SK orIK channel protein under immunologically reactive conditions, and thepresence of the bound antibody is detected directly or indirectly.

[0204] A. Non-Competitive Assay Formats

[0205] Immunoassays for detecting SK or IK channel proteins of thepresent invention include competitive and noncompetitive formats.Noncompetitive immunoassays are assays in which the amount of capturedanalyte (in this case an SK or IK channel protein) is directly measured.In one preferred “sandwich” assay, for example, the capture agent(anti-SK or anti-IK channel protein antibodies) can be bound directly toa solid substrate where they are immobilized. These immobilizedantibodies then capture SK or IK channel protein present in the testsample. The SK or IK channel protein thus immobilized is then bound by alabeling agent, such as a second human SK or IK channel protein antibodybearing a label. Alternatively, the second SK or IK channel proteinantibody may lack a label, but it may, in turn, be bound by a labeledthird antibody specific to antibodies of the species from which thesecond antibody is derived. The second can be modified with a detectablemoiety, such as biotin, to which a third labeled molecule canspecifically bind, such as enzyme-labeled streptavidin.

[0206] B. Competitive Assay Formats

[0207] In competitive assays, the amount of analyte (SK or IK channelprotein) present in the sample is measured indirectly by measuring theamount of an added (exogenous) analyte (SK or IK channel protein)displaced (or competed away) from a capture agent (anti-SK or anti-IKchannel protein antibody) by the analyte present in the sample. In onecompetitive assay, a known amount of, in this case, SK or IK channelprotein is added to the sample and the sample is then contacted with acapture agent, in this case an antibody that specifically binds the SKor IK channel protein. The amount of SK or IK channel protein bound tothe antibody is inversely proportional to the concentration of SK or IKchannel protein present in the sample.

[0208] In a particularly preferred embodiment, the antibody isimmobilized on a solid substrate. The amount of SK or IK channel proteinbound to the antibody may be determined either by measuring the amountof SK or IK channel protein present in the corresponding SK or IKchannel protein/antibody complex, or alternatively by measuring theamount of remaining uncomplexed SK or IK channel protein. The amount ofSK or IK channel protein may be detected by providing a labeled SK or IKchannel protein molecule.

[0209] A hapten inhibition assay is another preferred competitive assay.In this assay a known analyte, in this case the SK or IK channel proteinis immobilized on a solid substrate. A known amount of anti-SK oranti-IK channel protein antibody, respectively, is added to the sample,and the sample is then contacted with the immobilized SK or IK channelprotein. In this case, the amount of anti-SK or anti-IK channel proteinantibody bound to the immobilized SK or IK channel protein is inverselyproportional to the amount of SK or IK channel protein present in thesample. Again the amount of immobilized antibody may be detected bydetecting either the immobilized fraction of antibody or the fraction ofthe antibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

[0210] C. Other Assay Formats

[0211] In a particularly preferred embodiment, Western blot (immunoblot)analysis is used to detect and quantify the presence of an SK or IKchannel protein in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind SK channel protein. The anti-SK or anti-IKchannel protein antibodies specifically bind to the SK or IK channelproteins, respectively, on the solid support. These antibodies may bedirectly labeled or alternatively may be subsequently detected usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to the anti-SK or anti-IK channel protein.

[0212] Other assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.(1986) Amer. Clin. Prod. Rev. 5:34-41).

[0213] D. Labels

[0214] The particular label or detectable group used in the assay is nota critical aspect of the invention, so long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, radioisotopic, electrical, optical or chemical means.Useful labels in the present invention include those used in labeling ofnucleic acids as discussed, supra.

[0215] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

[0216] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to an anti-ligand (e.g., streptavidin)molecule which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

[0217] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904).

[0218] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0219] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

Immunoassay Detection Kits

[0220] The present invention also provides for kits for the diagnosis oforganisms (e.g., patients) with a deficiency in the levels of expressedSK or IK channel protein. The kits preferably include one or morereagents for detecting an the amount of SK or IK channel protein in amammal. Preferred reagents include antibodies that specifically bind tonormal SK or IK channel proteins or subsequences thereof. The antibodymay be free or immobilized on a solid support such as a test tube, amicrowell plate, a dipstick and the like. The kit may also containinstructional materials teaching the use of the antibody in an assay forthe detection of SK or IK channel protein. The kit may containappropriate reagents for detection of labels, positive and negativecontrols, washing solutions, dilution buffers and the like.

Assays for Compounds that Increase or Decrease K⁺ Flux

[0221] Isolated SK or IK channel nucleic acids of the present inventionwhich are expressed in cells can be used in a variety of assays todetect compounds that increase or decrease the flux (i.e., influx orefflux) of potassium through the SK or IK channels, respectively.Generally, compounds that decrease potassium ion flux will cause adecrease by at least 10% or 20%, more preferably by at least 30%, 40%,or 50%, and most preferably by at least 70%, 80%, 90% or 100%. Compoundsthat increase the flux of potassium ions will cause a detectableincrease in the potassium ion current density by increasing theprobability of a SK or IK channel being open and allowing the passage ofpotassium ions. Typically the flux will increase by at least 20%, 50%,100%, or 200%, often by at least 400%, 600%, 1,000%, 5,000% or 10,000%.Increased or decreased flux of potassium may be assessed by determiningchanges in polarization (i.e., electrical potential) of the cellexpressing the SK or IK channel. A particularly preferred means todetermine changes in cellular polarization is the voltage-clamptechnique. Whole cell currents are conveniently determined using theconditions set forth in Example 3. Other known assays include:radiolabeled rubidium-flux assays and fluorescence assays usingvoltage-ser.sitive dyes. See, e.g., Vestergarrd-Bogind et al., J.Membrane Biol., 88:67-75 (1988); Daniel et al., J. Pharmacol. Meth.,25:185-193 (1991); Holevinsky et al., J. Membrane Biology, 137:59-70(1994). Assays for compounds capable of inhibiting or increasingpotassium flux through the SK channel protein can be performed byapplication of the compounds to a bath solution in contact with andcomprising cells having an SK or IK channel of the present invention.See, e.g., Blatz et al., Nature, 323:718-720 (1986); Park, J. Physiol.,481:555-570 (1994). Generally, the compounds to be tested are present inthe range from 1 pM to 100 mM. Changes in function of the channels canbe measured in the electrical currents or ionic flux, or by theconsequences of changes in currents and flux.

[0222] The effects of the test compounds upon the function of thechannels can be measured by changes in the electrical currents or ionicflux or by the consequences of changes in currents and flux. Changes inelectrical current or ionic flux are measured by either increases ordecreases in flux of cations such as potassium or rubidium ions. Thecations can be measured in a variety of standard ways. They can bemeasured directly by concentration changes of the ions or indirectly bymembrane potential or by radiolabeling of the ions. Consequences of thetest compound on ion flux can be quite varied. Accordingly, any suitablephysiological change can be used to assess the influence of a testcompound on the channels of this invention. Changes in channel functioncan be measured by ligand displacement such as CTX release. When thefunctional consequences are determined using intact cells or animals,one can also measure a variety of effects such as transmitter release(eg., dopamine), hormone release (e.g., insulin), transcriptionalchanges to both known and uncharacterized genetic markers (e.g.,northern blots), cell volume changes (e.g., in red blood cells),immuno-responses (e.g., T cell activation), changes in cell metabolismsuch as cell growth or pH changes.

[0223] Preferably, the SK channel of the assay will be selected from achannel protein of SEQ ID NOS: 1, 2, 3, 4, 19, 20, 43 or 47 orconservatively modified variant thereof. An IK channel of the assay willpreferably have a sequence as shown in SEQ ID NO:32, or conservativelymodified variant thereof. Alternatively, the SK channel of the assaywill be derived from a eukaryote and include an amino acid subsequencehaving sequence similarity to the core region of SK channel proteins ofSEQ ID NOS:1 through 4, 19, 20, 43 and/or 47. The IK will typically bederived from a eukaryote and include an amino acid subsequence havingsequence similarity to the core region of IK channel proteins of SEQ IDNO:32. Generally, the functional SK or IK channel protein will be atleast 400, 450, 500, or 550 amino acids in length. The percentage ofsequence-similarity with the core region of a protein selected from thegroup consisting of: SEQ ID NO:1, 2, 3, 4, 19, 20, 32, 43 and 47 will beany one of the integers between 60 and 100. Generally, the sequencesimilarity will be at least 60%, typically at least 70%, generally atleast 75%, preferably at least 80%, more preferably at least 85%, mostpreferably at least 90%, and often at least 95%. Thus, SK channelhomologs will hybridize, under moderate hybridization conditions, to anucleic acid of at least 300 nucleotides in length from the core regionof a nucleic acid selected from the group consisting of SEQ ID NOS:13,14, 15, 16, 21, 22, and complementary sequences thereof. IK channelhomologs will hybridize, under moderate hybridization conditions, to anucleic acid of at least 300 nucleotides in length from the core regionof a nucleic acid such as SEQ ID NO:31.

[0224] The “core region” or “core sequence” of SEQ ID NOS:13-16, 21,22,44 and 48 corresponds to the encoded region of alignment between SEQID NOS: 1, 2, 3, 4, 19, 20, 43, and 47 with and from rSK2 (SEQ ID NO:2)amino acid residue 135 to 462. The core region of hIK1 is from aminoacid residue 25 through residue 351. In preferred embodiments, the SKchannel will have at least 90% sequence similarity, as compared to thecore sequence from a sequence of ID NO:1, 2, 3, 4, 19, 20, 43, or 47over a comparison window of any of from any one of 20 contiguous aminoacid residues to 300 contiguous amino acid residues from within the coreregion. In preferred embodiments, the IK channel will have at least 90%sequence similarity, as compared to the core sequence of SEQ ID NO:32,over a comparison window of any of from any one of 20 contiguous aminoacid residues to 300 contiguous amino acid residues from within the coreregion.

[0225] The SK channel homologs will generally have substantially similarconductance characteristics (e.g., 2-60 pS) and calcium sensitivities(30 nM-10 μM). IK channel homologs will likewise have similar SKchannels conductance characteristics as a IK channel (e.g., 20-80 pS)and calcium sensitivities (30 nM-10 μM). Chimeras formed by expressionof at least two of SEQ ID NOS: 1, 2, 3, 4, 19, 20, or 32 can also beused. In a preferred embodiment, the cell placed in contact with acompound which is assayed for increasing or decreasing potassium flux isa eukaryotic cell, more preferably an oocyte of Xenopus (e.g., Xenopuslaevis).

[0226] Yet another assay for compounds that increase or decreasepotassium flux in calcium activated potassium channels involves “virtualgenetics,” in which a computer system is used to generate athree-dimensional structure of SK and IK proteins based on thestructural information encoded by the amino acid sequence. The aminoacid sequence interacts directly and actively with a preestablishedalgorithm in a computer program to yield secondary, tertiary, andquaternary structural models of the protein. The models of the proteinstructure are then examined to identify regions of the structure thathave the ability to bind to ligands. These regions are then used toidentify ligands that bind to the protein.

[0227] The three-dimensional structural model of the protein isgenerated by inputting channel protein amino acid sequences or nucleicacid sequences encoding a channel protein into the computer system. Theamino acid sequence of the channel protein is selected from the groupconsisting of: SEQ ID NOS:1, 2, 3, 4, 19, 20, 32, 43, 47, andconservatively modified versions thereof. The amino acid sequencerepresents the primary sequence of the protein, which encodes thestructural information of the protein. The amino acid sequence is inputinto the computer system from computer readable substrates that include,but are not limited to, electronic storage media (e.g., magneticdiskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM,telephone lines), addresses to internet sites, and RAM. Thethree-dimensional structural model of the channel protein is thengenerated by the interaction of the amino acid sequence and the computersystem. The software is commercially available programs such asBiopolymer, Quanta, and Insight.

[0228] The amino acid sequence represents a primary structure thatencodes the information necessary to form the secondary, tertiary andquaternary structure of the protein. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potential, hydrohobic potential, solventaccessible surface, and hydrogen bonding. Secondary energy terms includevan der Waals potential. Biological molecules form the structures thatminimize the energy terms in a cumulative fashion. The computer programis therefore using these terms encoded by the primary structure or aminoacid sequence to create the secondary structural model.

[0229] The tertiary structure of the protein encoded by the secondarystructure is then formed on the basis of the energy terms of thesecondary structure. The user at this point can input additionalvariables such as whether the protein is membrane bound or soluble, itslocation in the body, and whether it is cytoplasmic, surface, ornuclear. These variables along with the energy terms of the secondarystructure are used to form the model of the teritary structure. Inmodeling the tertiary structure, the computer program matcheshydrophobic protein faces of secondary structure with like, andhydrophilic secondary structure with like.

[0230] Finally, quaternary structure of multi-subunit proteins can bemodeled in a similar fashion, using anisotrophy terms. These termsinterface different protein subunits to energetically minimize theinteraction of the subunits. In the case of channel proteins, typicallyfour identical subunits make up the quaternary structure of the channel.

[0231] Once the structure has been generated, potential ligand bindingregions are identified by the computer system. Three-dimensionalstructures for potential ligands are generated by inputting amino acidand nucleotide sequences or chemical formulas of compounds, as describedabove. The three-dimensional structure of the potential ligand is thencompared to that of the channel protein to identify ligands that bind tothe channel protein. Binding affinity between the protein and ligands isdetermined using energy terms to determine which ligands have anenhanced probability of binding to the protein.

[0232] Computer systems are also used to screen for mutations of SK andIK genes. Such mutations can be associated with disease states. Once themutations are identified, diagnostic assays can be used to identifypatients having such mutated genes associated with disease states.Identification of the mutated SK and IK genes involves receiving inputof a first nucleic acid sequence encoding a calcium channel proteinhaving an amino acid sequence selected from the group consisting of SEQID NOS:1, 2, 3, 4, 20, 32, 43, 47, and conservatively modified versionsthereof. The sequence is input into the computer system as describedabove. The first nucleic acid sequence is then compared to a secondnucleic acid sequence that has substantial identity to the first nucleicacid sequence. The second nucleic acid sequence is input into thecomputer system in the manner described above. Once the first andsequence sequences are compared, nucleotide differences between thesequences are identified. Such sequences can represent allelicdifferences in SK and IK genes, and mutations associated with diseasestates.

Cellular Transfection and Gene Therapy

[0233] The present invention provides packageable SK and IK channelprotein nucleic acids (cDNAs), supra, for the transfection of cells invitro and in vivo. These packageable nucleic acids can be inserted intoany of a number of well known vectors for the transfection of targetcells and organisms as described below. The nucleic acids aretransfected into cells, ex vivo or in vivo, through the interaction ofthe vector and the target cell. The SK or IK channel protein nucleicacid, under the control of a promoter, then expresses thecalcium-activated potassium channel protein of the present inventionthereby mitigating the effects of absent, partial inactivation, orabnormal expression of the SK or IK channel protein gene.

[0234] Such gene therapy procedures have been used to correct acquiredand inherited genetic defects, cancer, and viral infection in a numberof contexts. The ability to express artificial genes in humansfacilitates the prevention and/or cure of many important human diseases,including many diseases which are not amenable to treatment by othertherapies. As an example, in vivo expression of cholesterol-regulatinggenes, genes which selectively block the replication of HIV, andtumor-suppressing genes in human patients dramatically improves thetreatment of heart disease, AIDS, and cancer, respectively. For a reviewof gene therapy procedures, see Anderson, Science (1992) 256:808-813;Nabel and Felgner (1993) TIBTECH 11: 211-217; Mitani and Caskey (1993)TIBTECH 11: 162-166; Mulligan (1993) Science 926-932; Dillon (1993)TIBTECH 11: 167-175; Miller (1992) Nature 357: 455-460; Van Brunt (1988)Biotechnology 6(10): 1149-1154; Vigne (1995) Restorative Neurology andNeuroscience 8: 35-36; Kremer and Perricaudet (1995) British MedicalBulletin 51(1) 31-44; Haddada et al. (1995) in Current Topics inMicrobiology and Immunology Doerfler and Böhm (eds) Springer-Verlag,Heidelberg Germany; and Yu et al., Gene Therapy (1994) 1:13-26.

[0235] Delivery of the gene or genetic material into the cell is thefirst critical step in gene therapy treatment of disease. A large numberof delivery methods are well known to those of skill in the art. Suchmethods include, for example liposome-based gene delivery (Debs and Zhu(1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques6(7): 682-691; Rose U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309;and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414), andreplication-defective retroviral vectors harboring a therapeuticpolynucleotide sequence as part of the retroviral genome (see, e.g.,Miller et al (1990) Mol. Cell. Biol. 10:4239 (1990); Kolberg (1992) J.NIH Res. 4:43, and Cometta et al. Hum. Gene Ther. 2:215 (1991)). Widelyused retroviral vectors include those based upon murine leukemia virus(MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus(SIV), human immuno deficiency virus (HIV), and combinations thereof.See, e.g., Buchscher et al. (1992) J. Virol. 66(5) 2731-2739; Johann etal. (1992) J. Virol. 66 (5):1635-1640 (1992); Sommerfelt et al., (1990)Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller etal., J. Virol. 65:2220-2224 (1991); Wong-Staal et al., PCT/US94/05700,and Rosenburg and Fauci (1993) in Fundamental Immunology, Third EditionPaul (ed) Raven Press, Ltd., New York and the references therein, and Yuet al., Gene Therapy (1994) supra).

[0236] AAV-based vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and in in vivo and ex vivo gene therapy procedures. See, Westet al. (1987) Virology 160:38-47; Carter et al. (1989) U.S. Pat. No.4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human GeneTherapy 5:793-801; Muzyczka (1994) J. Clin. Invst. 94:1351 and Samulski(supra) for an overview of AAV vectors. Construction of recombinant AAVvectors are described in a number of publications, including Lebkowski,U.S. Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol.5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol., 4:2072-2081;Herrnonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, 81:6466-6470;McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol.,63:03822-3828. Cell lines that can be transfected by rAAV include thosedescribed in Lebkowski et al. (1988) Mol. Cell. Biol., 8:3988-3996.

[0237] A. Ex vivo Transfection of Cells

[0238] Ex vivo cell transfection for diagnostics, research, or for genetherapy (e.g., via re-infusion of the transfected cells into the hostorganism) is well known to those of skill in the art. In a preferredembodiment, cells are isolated from the subject organism, transfectedwith an SK or IK channel protein nucleic acid (gene or cDNA), andre-infused back into the subject organism (e.g., patient). Various celltypes suitable for ex vivo transfection are well known to those of skillin the art (see, e.g., Freshney et al., Culture of Animal Cells, aManual of Basic Technique, third edition Wiley-Liss, New York (1994))and the references cited therein for a discussion of how to isolate andculture cells from patients).

[0239] As indicated above, in a preferred embodiment, the packageablenucleic acid which encodes an SK or IK channel protein is under thecontrol of an activated or constitutive promoter. The transfectedcell(s) express a functional SK or IK channel protein which mitigatesthe effects of deficient or abnormal SK or IK channel protein geneexpression.

[0240] In one particularly preferred embodiment, stem cells are used inex-vivo procedures for cell transfection and gene therapy. The advantageto using stem cells is that they can be differentiated into other celltypes in vitro, or can be introduced into a mammal (such as the donor ofthe cells) where they will engraft in the bone marrow. Methods fordifferentiating CD34⁺ cells in vitro into clinically important immunecell types using cytokines such a GM-CSF, IFN-γ and TNF-α are known(see, Inaba et al. (1992) J. Exp. Med. 176, 1693-1702, and Szabolcs etal. (1995) 154: 5851-5861).

[0241] Stem cells are isolated for transduction and differentiationusing known methods. For example, in mice, bone marrow cells areisolated by sacrificing the mouse and cutting the leg bones with a pairof scissors. Stem cells are isolated from bone marrow cells by panningthe bone marrow cells with antibodies which bind unwanted cells, such asCD4⁺ and CD8⁺ (T cells), CD45⁺ (panB cells), GR-1 (granulocytes), andlad (differentiated antigen presenting cells). For an example of thisprotocol see, Inaba et al. (1992) J. Exp. Med: 176, 1693-1702.

[0242] In humans, bone marrow aspirations from iliac crests areperformed e.g., under general anesthesia in the operating room. The bonemarrow aspirations is approximately 1,000 ml in quantity and iscollected from the posterior iliac bones and crests. If the total numberof cells collected is less than about 2×10⁸/kg, a second aspirationusing the sternum and anterior iliac crests in addition to posteriorcrests is performed. During the operation, two units of irradiatedpacked red cells are administered to replace the volume of marrow takenby the aspiration. Human hematopoietic progenitor and stem cells arecharacterized by the presence of a CD34 surface membrane antigen. Thisantigen is used for purification, e.g., on affinity columns which bindCD34. After the bone marrow is harvested, the mononuclear cells areseparated from the other components by means of ficol gradientcentrifugation. This is performed by a semi-automated method using acell separator (e.g., a Baxter Fenwal CS3000+ or Terumo machine). Thelight density cells, composed mostly of mononuclear cells are collectedand the cells are incubated in plastic flasks at 37° C. for 1.5 hours.The adherent cells (monocytes, macrophages and B-Cells) are discarded.The non-adherent cells are then collected and incubated with amonoclonal anti-CD34 antibody (e.g., the murine antibody 9C5) at 4° C.for 30 minutes with gentle rotation. The final concentration for theanti-CD34 antibody is 10 μg/ml. After two washes, paramagneticmicrospheres (Dyna Beads, supplied by Baxter Immunotherapy Group, SantaAna, Calif.) coated with sheep antimouse IgG (Fc) antibody are added tothe cell suspension at a ratio of 2 cells/bead. After a furtherincubation period of 30 minutes at 4° C., the rosetted cells withmagnetic beads are collected with a magnet. Chymopapain (supplied byBaxter Immunotherapy Group, Santa Ana, Calif.) at a final concentrationof 200 U/ml is added to release the beads from the CD34+ cells.Alternatively, and preferably, an affinity column isolation procedurecan be used which binds to CD34, or to antibodies bound to CD34 (see,the examples below). See, Ho et al. (1995) Stem Cells 13 (suppl. 3):100-105. See also, Brenner (1993) Journal of Hematotherapy 2: 7-17.

[0243] In another embodiment, hematopoetic stem cells are isolated fromfetal cord blood. Yu et al. (1995) Proc. Natl. Acad. Sci. USA, 92:699-703 describe a preferred method of transducing CD34⁺ cells fromhuman fetal cord blood using retroviral vectors.

[0244] B. In vivo Transfection

[0245] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)containing therapeutic nucleic acids can be administered directly to theorganism for transduction of cells in vivo. Administration is by any ofthe routes normally used for introducing a molecule into ultimatecontact with blood or tissue cells. The packaged nucleic acids areadministered in any suitable manner, preferably with pharmaceuticallyacceptable carriers. Suitable methods of administering such packagednucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

[0246] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention.

[0247] Formulations suitable for oral administration can consist of (a)liquid solutions, such as an effective amount of the packaged nucleicacid suspended in diluents, such as water, saline or PEG 400; (b)capsules, sachets or tablets, each containing a predetermined amount ofthe active ingredient, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of lactose, sucrose, mannitol, sorbitol,calcium phosphates, corn starch, potato starch, tragacanth,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, fillers, binders, diluents, buffering agents,moistening agents, preservatives, flavoring agents, dyes, disintegratingagents, and pharmaceutically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastiles comprising the active ingredient inan inert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

[0248] The packaged nucleic acids, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

[0249] Suitable formulations for rectal administration include, forexample, suppositories, which consist of the packaged nucleic acid witha suppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the packaged nucleic acid with a base, including, forexample, liquid triglycerides, polyethylene glycols, andparaffin-hydrocarbons.

[0250] Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradernal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

[0251] Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by the packaged nucleic acid as described above in thecontext of ex vivo therapy can also be administered intravenously orparenterally as described above.

[0252] The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

[0253] In determining the effective amount of the vector to beadministered in the treatment or prophylaxis of conditions owing todiminished or aberrant expression of SK or IK channel protein, thephysician evaluates circulating plasma levels of the vector, vectortoxicities, progression of the disease, and the production ofanti-vector antibodies. In general, the dose equivalent of a nakednucleic acid from a vector is from about 1 μg to 100 μg for a typical 70kilogram patient, and doses of vectors which include a retroviralparticle are calculated to yield an equivalent amount of therapeuticnucleic acid.

[0254] For administration, inhibitors and transduced cells of thepresent invention can be administered at a rate determined by the LD-50of the inhibitor, vector, or transduced cell type, and the side-effectsof the inhibitor, vector or cell type at various concentrations, asapplied to the mass and overall health of the patient. Administrationcan be accomplished via single or divided doses.

[0255] In a preferred embodiment, prior to infusion, blood samples areobtained and saved for analysis. Between 1×10⁸ and 1×10¹² transducedcells are infused intravenously over 60- 200 minutes. Vital signs andoxygen saturation by pulse oximetry are closely monitored. Blood samplesare obtained 5 minutes and 1 hour following infusion and saved forsubsequent analysis. Leukopheresis, transduction and reinfusion can berepeated are repeated every 2 to 3 months. After the first treatment,infusions can be performed on a outpatient basis at the discretion ofthe clinician. If the reinfusion is given as an outpatient, theparticipant is monitored for at least 4, and preferably 8 hoursfollowing the therapy.

[0256] Transduced cells are prepared for reinfusion according toestablished methods. See, Abrahamsen et al. (1991) J. Clin. Apheresis,6: 48-53; Carter et al. (1988) J. Clin. Arpheresis, 4:113-117; Aebersoldet al. (1988) J. Immunol. Meth., 112: 1-7; Muul et al. (1987) J.Immunol. Methods, 101:171-181 and Carter et al. (1987) Transfusion 27:362-365. After a period of about 2-4 weeks in culture, the cells shouldnumber between 1×10⁸ and 1×10¹². In this regard, the growthcharacteristics of cells vary from patient to patient and from cell typeto cell type. About 72 hours prior to reinfusion of the transducedcells, an aliquot is taken for analysis of phenotype, and percentage ofcells expressing the therapeutic agent.

[0257] Although the present invention has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

EXAMPLE 1

[0258] Example 1 describes the isolation and sequencing of clonesencoding small and intermediate conductance, calcium-dependent potassiumchannels.

[0259] A. Small conductance potassium channels, with the exception ofthe minK protein (Takumi et al., Science, 242:1042-1045 (1988), share acommon structural motif within the pore region including the sequencewhich dictates the characteristic selectivity sequence for monovalentcations (Heginbotham et al., Biophys. J., 66:1061-1067 (1994)).

[0260] A BLAST search of the EST database using the query sequenceFXSIPXXXWWAXVTMTTVGYGDMXP (SEQ ID NO:45), allowing for mismatches,retrieved known potassium-channel-sequences-and Genbank #M62043.Oligonucleotides corresponding to nucleotides 6-36 (sense) and 258-287(antisense) of #M62043 were synthesized (Genosys, The Woodlands, Tex.),radiolabeled using polynucleotide kinase (BRL) and ³²P-ATP (NEN), andused to screen ˜10⁶ recombinant phage from the human hippocampal cDNAlibrary (40% formamide; 1 M NaCl, 1% SDS, 37° C.; washed at 1×SSC, 50°C.). Double positively hybridizing phage were purified by rescreening atreduced densities. cDNA inserts were subcloned into M13 and thenucleotide sequences determined using the dideoxy chain terminationmethod and T7 DNA polymerase (Sequenase, UBI). A fragment of this clonecontaining the pore domain (amino acids 325-522) was radiolabeled usingrandom primers (Boehringer) and used to screen a rat brain cDNA library(30% formamide, 1 M NaCl, 1% SDS, 37 ° C.; washed at 2×SSC, 50° C.).Positively hybridizing phage were purified and the nucleotide sequencesof the inserts determined. Computer analyses were performed using theGCG software suite (Genetics Computer Group; version 8.1).

[0261] In addition to known potassium channels, one of the detectedsequences from human hippocampus suggested it may contain the consensusmotif, but included several ambiguities (Genbank #M62043). Based uponthis sequence, oligonucleotides were synthesized having the sequencerepresented by nucleotides 6 to 36 of the sense strand; and nucleotides258 to 287 of the antisense strand. The oligonucleotides were used toprobe a human hippocampal cDNA library.

[0262] A full length coding sequence, hSKI (SEQ ID NO:13), was isolatedand analyzed for open reading frames, Kozak consensus sequences,potential transmembrane domains, and predicted protein structure. Afragment containing the putative pore region was radiolabelled by randompriming and subsequently used to probe a rat brain cDNA library using ahybridization solution of 40% formamide, 1 M NaCl, 1% SDS, and 100 μg/mlyeast RNA, at 37° C. and washed using 0.5×SSC at 55° C. Two clonescontaining different full length coding sequences were isolated andanalyzed: rSK2 (SEQ ID NO:15), and rSK3 (SEQ ID NO:16). In addition, apartial clone was identified representing the rat homolog of hSK1 (rSK1(SEQ ID NO:14)).

[0263] The sequences predict proteins of 561 amino acids for hSK1 (SEQID NO:1), 580 amino acids for rSK2 (SEQ ID NO:2), and 553 amino acidsfor rSK3 (SEQ ID NO:3) which contain no stretches of homology (i.e., nosignal above background under low stringency conditions) with othercloned potassium channels apart from a 12 amino acid sequence in theputative pore region. Hydrophobicity analysis predicts six transmembranesegments with the N- and C-termini residing inside the cell. Thesequences are highly conserved across their transmembrane cores (80-90%identity), but diverge in sequence and length within their N- andC-terminal domains (Table 1). TABLE 1 rSK2 .......... ........MSSCRYNGGVMR PLSNLSSSRR                                  NLHEMDSEAQ rSK3.......... ........MS SCKYSGGVMK PLSRLSASRR                                 NLTEAEPEGQ rSK1 .......... .................... ..........                                  .......... hSK1MPGPRAACSE PNPCTQVVMN SHSYNGSVGR P...LGSGPG                                 ALGRDPPDPE rSK2 PLQPPASVVG GGGGASSPSAAAAASSSAPE IVVSKPEHNN                                  SNNLALYGTG rSK3PLQLF..... .......... ...SPSNPPE IIISSREDNH                                 AHQTLLHHPN rSK1 .......... .................... ..........                                  .......... hSK1AGHPPQPPHS PGLQVVVAKS EPARPSPGSP RGQPQDQDDD                                 EDDEEDEAGR rSK2 GGGSTGGGGG GGGGGGGSGHGSSSGTKSSK KKNQNIGYKL                                  GHRRALFEKR rSK3ATHNHQHAGT TAGSTTFP.. ......KANK RKNQNIGYKL                                 GHRRALFEKR rSK1 .......... ...................S GKPPTVSHRL                                  GHRRALFEKR hSK1QR........ .......... ........AS GKPSNVGHRL                                 GHRRALFEKR rSK2 KRLSDYALIF GMFGTVVMVIETELSWGAYD KASLYSLALK                                  CLISLSTIIL rSK3KRLSDYALIF GMFGTVVMVI ETELSWGLYS KDSMFSLALK                                 CLISLSTIIL rSK1 KRLSDYALIF GMFGIVVMVTETELSWGVYT KESLCSFALK                                  CLISLSTVIL hSK1KRLSDYALTF GMFGTVVMVT ETELSWGVYT KESLYSFALK                                 CLISLSTAIL rSK2 LGLIIVYHAR EIQLFMVDNGADDWRIANTY ERIFFICLEI                                  LVCAIHPIPG rSK3LGLIIAYHTR EVQLFVIDNG ADDWRTAMTY ERILYISLEM                                 LVCAIHPTPG rSK1 LGLVILYHAR EIQLFLVDNGADDWRTAMTW ERVSLISLEL                                  AVCATHPVPG hSK1LGLVVLYHAR ETQLFMVDNG ADDWRIAMTC ERVFLISLEL                                 AVCAIHPVPG rSK2 NYTFTWTARL AFSYAPSTTTADVDTTLSTP MFLRLYLIAR                                  VMLLHSKLFT rSK3EYKFFWTARL AFSYTPSRAE ADVDTTLSTP MFLRLYLIAR                                 VMLLHSKLFT rSK1 HYRFTWTARL APSLVPSAAEADVDVLLSTP MFLRLYLLAR                                  VMLLHSRIFT hSK1HYRFTWTARL AFTYAPSVAE ADVDVLLSTP MFLRLYLLGR                                 VMLLHSKIET rSK2 DASSRSIGAL NKINFNTRFVMKTLMTICPG TVLLVFSISL                                  WTIAAWTVRA rSK3DASSRSTGAL NKINFNTRFV MKTLMTICPG TVLLMFSTSL                                 WIIAAWTVRV rSK1 DASSRSIGAL NRVTFNTRFVTKTLMTTCPG TVLLVFSISS                                  WIVAAWTVRV hSK1DASSRSIGAL NKITFNTRFV MKTLMTICPG TVLLVFSISS                                 WIIAAWTVRV rSK2 CERYHDQQDV TSNFLGAMWLISITFLSIGY GDMVPNTYCG                                  KGVCLLTGIM rSK3CERYHDQQDV TSNFLGAMWL ISITFLSIGY GDMVPHTYCG                                 KGVCLLTGIM rSK1 CERYHDKQEV TSNFLGAMWLISITFLSIGY GDMVPHTYCG                                  KGVCLLTGIM hSK1CERYHDKQEV TSNFLGAMWL ISITELSIGY GDMVPHTYCG                                 KGVCLLTGIM rSK2 GAGCTALVVA VVARKLELTKAEKHVHNFMM DTQLTKRVKN                                  AAANVLRETW rSK3GAGCTALVVA VVARKLELTK AEKHVHNFMM DTQLTKRIKN                                 AAANVLRETW rSK1 GAGCTALVVA VVARKLELTKAEKHVHNFMM DTQLTKRVKN                                  AAANVLRETW hSK1GAGCTALVVA VVARKLELTK AEKHVHNFMM DTQLTKRVKN                                 AAANVLRETW rSK2 LIYKNTKLVK KIDHAKVRKHQRKFLQAIHQ ...LRSVKME                                  QRKLNDQANT rSK3LIYKHTKLLK KIDHAKVRKH QRKFLQAIHQ ...LRGVKME                                 QRKLSDQANT rSK1 LIYKHTRLVK KPDQSRVRKHQRKFLQAIHQ AQKLRTVKIE                                  QGKVNDQANT hSK1LIYKHTRLVK KPDQARVRKH QRKFLQATHQ AQKLRSVKIE                                 QGKLNDQANT rSK2 LVDLAKTQNI MYDMISDLNERSEDFEKRIV TLETKLETLI                                  GSIHALPGLI rSK3LVDLSKMQNV MYDLITELND RSEDLEKQIG SLESKLEHLT                                 ASFNSLPLLI rSK1 LADLAKAQSI AYEVVSELQAQQEELEARLA ALESRLDVLG                                  ASLQALPSLI hSK1LTDLAKTQTV MYDLVSELHA QHEELEARLA TLESRLDALG                                 ASLQALPGLI rSK2 SQTI....RQ QQRDFIETQMENYDKHVTYN AERSRSSSRR                                  RRSSSTAPPT rSK3ADTLRQQQQQ LLTAFVEARG ISVAVG.... ..........                                 ...TSHAPPS rSK1 AQAICPLPPP W...PGPSHLTTAAQSPQSH WLPTTASDCG                                  *.........

[0264] The fourth predicted membrane spanning domain contains 3positively charged residues that do not occupy every third position asin voltage-dependent potassium channels (Durell et al., Biophys. J.,62:238-250 (1992)), but are separated by 6 and 7 residues. There aremultiple consensus targets for phosphorylation by a variety of proteinkinases. Some of these sites are found in all clones. However, eachclone contains potential phosphorylation sites not conserved among allmembers. There are no conserved N-linked glycosylation sites (NXXS/T)(SEQ ID NO:46) in predicted extracellular domains, and no consensusnucleotide or calcium binding domains (E-F hands).

[0265] Northern blots of rat brain and skeletal muscle showed that rSK3transcripts from these tissues encoded proteins that were N-terminallyextended relative to the rSK3 clone SEQ ID NO:16. The nucleic acidencoding the rSK3 N-terminal extension was cloned and sequenced, and thecDNA encoding N-terminal extended rSK3 is represented by SEQ ID NO:44.In addition, endogenous rSK3 was shown to have a nucleotide sequencethat encodes a protein having a C-terminus with the last 5 amino acidsof SEQ ID NO:3 replaced by the last 9 amino acids of SEQ ID NO:43.Similarly, hSK3 was shown to have an N-terminal extension, and the cDNAencoding this N-terminal extension is represented by SEQ ID NO:48.

[0266] B. To isolate intermediate conductance calcium activated K⁺proteins, one can use PCR under standard conditions. Suitable primersare SEQ ID NOS:34 and 35 which yield a probe of about 270 bases and SEQID NOS:36 and 37 which yield a probe of about 165 bases. These primerscan be used to amplify plasmid DNA comprising cloned hIK1 or on reversetranscribed RNA from a tissue which expresses hIK1, such as a cDNAlibrary from pancreas. The PCR reaction will yield DNA fragments of thespecified size which contain sequences specific to hIK1 and relatedgenes. These DNA fragments are subsequently labeled for useas-hybridization probes-by standard random-priming protocols. Thelabeled probes are then used to screen libraries at high stringency toisolate only hIK1 sequences, or at moderately low stringency (30-40%formamide, 37° C. hyb/ 1×SSC, 55° C. wash) to isolate putatively relatedsequences. Alternatively, one can amplify the intact hIK1 gene from apancreas cDNA library using PCR primer pair SEQ ID NOS:38 and 39 or 40and 41.

EXAMPLE 2

[0267] Example 2 describes in situ hybridization of rat brain sectionsusing sequences distinct for each of the rat SK channel clones, anddetermination of transcript sizes from various peripheral tissues.

[0268] Care and handling of adult female Sprague-Dawley rats were inaccordance with the highest standards of institutional guidelines. Ratswere deeply anesthetized with pentobarbital and perfused transcardiallywith ice-cold saline, followed by ice-cold 4% paraformaldehyde in 0.1 Msodium borate (pH 9.5). The brains were removed quickly and post-fixedovernight at 4 ° C. in 4% paraformaldehyde in borate buffer (pH 9.5)containing 10% sucrose. Cryostat microtome sections (25 mm) were mountedonto gelatin- and poly-L-lysine-coated glass slides and incubated for 15min in 4% paraformaldehyde in 0.1 M PBS, washed twice in 0.1 M PBS, andtreated for 30 min at 37° C. in 10 mg/ml proteinase K in 100 mM Tris, 50mM EDTA (pH 8), followed by 0.0025% acetic anhydride in 0.1 Mtriethanolamine at room temperature. The sections were then washed in2×SSC, dehydrated in increasing concentrations of ethanol, andvacuum-dried at room temperature.

[0269] Templates for probe synthesis represented C-terminal and 3′untranslated sequences unique to each of the clones, and were subclonedinto pKS. Using linearized template DNA, ³⁵S -labeled antisense cRNAprobe heated to 65 ° C. for 5 min and diluted to 10⁷ cpm/ml inhybridization buffer; 66 % formamide, 260 mM NaCl, 1.3×Denhardtsolution, (13 mM Tris, pH 8.0,1.3 mM EDTA, 13 % dextran sulfate).Sections in hybridization mixture were covered with siliconized glasscoverslips and sealed using DPX mountant. After incubating at 58 ° C.for 20 hr, the slides were soaked in 4×SSC to remove coverslips, thenrinsed in 4×SSC (4 times, 5 min each) prior to ribonuclease A treatment(20 mg/ml for 30 min at 37 ° C.). The slides were then rinsed indecreasing concentrations of SSC containing 1 mM DTT to a finalstringency of 0.1×SSC, 1 mM DTT for 30 min at 65° C. After dehydratingthe sections in increasing concentrations of ethanol, they werevacuum-dried and exposed to DuPont Cronex-4 X-ray film for 7 days. Thefilm was scanned by a Microtek ScanMaker 1850S at 728pixel/cm-resolution and the images analyzed using image v1.55 -software(NIH) and Photoshop (Adobe).

[0270] The results indicate that mRNAs to the rat sequences are broadlydistributed throughout the CNS, in characteristic but overlappingpatterns. rSK1 is expressed in the hippocampus and the dentate gyrus,the granular layer of the cerebellum, and the anterior olfactorynucleus. rSK1 mRNA was also detected in the subiculum, the olfactorytubercle, and the neocortex. rSK2 mRNA is the most widely expressed,with highest expression in the hippocampus and lower levels in thedentate gyrus, the olfactory bulb and the anterior olfactory nucleus.rSK2 mRNA was also detected in the granular layer of the cerebellum, thereticular nucleus of the thalamus, and the pontine nucleus. The patternof in situ hybridization for rSK2 mRNA is coincident with the pattern ofradiolabeled apamin binding in rat brain (Gelhart, Neuroscience,52:191-205 (1993)). rSK3 mRNA was detected in the olfactory tubercle andolfactory bulb, throughout the thalamus, the lateral septum, the ventraltegmental area, and the substantia nigra pars compacta. Moderate levelswere detected throughout the hypothalamus, the caudate putamen, and thenucleus accumbens.

[0271] The same distinct sequences for rSK1 and rSK2 were used to probeNorthern blots prepared with mRNA isolated from total brain and severalperipheral tissues. Total RNA was extracted (Chirgwin et al., Biochem.,18:5294-5300 (1979)) from rat brain, adrenal gland, thymus, spleen,skeletal muscle, heart, kidney, liver, and lung of 3 week oldSprague-Dawley rats. Poly (A)⁺ mRNA was purified by oligo d(T) cellulosechromatography (Collaborative Research), and 3 μg from each tissue wasprepared as a Northern blot by electrophoresis through a 1%agarose-formaldehyde gel and transfer to Genescreen (NEN) nylonmembranes. Antisense riboprobes of the same sequence as used for in situhybridization were synthesized from linearized DNA templates using³²P-UTP (NEN). Blots were hybridized in 50% formamide, 5% SDS, 400 mMNaPO₄, 1 mM EDTA at 60° C. for 12 hours, followed by washes in 0.05×SSCat 65° C., and visualized using a Phosphorimager 445 SI (MolecularDynamics) after 15 hours.

[0272] rSK1 mRNA was detected in rat brain and heart, while rSK2 mRNAwas detected in brain and adrenal gland. The results show that rSK1mRNAs of different sizes are present in brain (3.2 kb) and heart (4.4kb). rSK2 mRNA was detected in brain and adrenal gland as two bands of2.2 and 2.4 kb. Neither rSK1 nor rSK2 mRNA was detected from lung,liver, kidney, thymus, spleen, or skeletal muscle.

EXAMPLE 3

[0273] Example 3 describes in vitro-expression of SK and IK channelproteins.

[0274] 3A. Example 3A describes in vitro expression of rSK2 and hSK1mRNAs in Xenopus oocytes and measurements of electrical conductance.

[0275] In vitro mRNA synthesis and oocyte injections were performed asdescribed in Adelman et al., Neuron, 9:209-216 (1992). Xenopus care andhandling were in accordance with the highest standards of institutionalguidelines. Frogs underwent no more than two surgeries, separated by atleast three weeks, and surgeries were performed using well establishedtechniques. Frogs were anesthetized with an aerated solution of3-aminobenzoic acid ethyl ester.

[0276] Oocytes were studied 2-5 days after injection with 2 ng of mRNA.Whole cell currents were measured after mRNA injection using a twoelectrode voltage clamp with a CA-1 amplifier interfaced to a MacintoshQuadra 650 computer. Data were simultaneously acquired through Pulse(Heka, Germany) at 500 Hz and Chart (AD Instruments, Australia) at 10Hz. During recording, oocytes were continuously superfused with ND-96solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂,5 mMHEPES (pH 7.5 with NaOH) at room temperature. To minimize Cl⁻ currents,some oocytes were soaked and studied in Cl⁻-free ND96 solution (96 mMNagluconate, 2 mM Kgluconate, 2.7 mM Cagluconate₂, 1 mM Mggluconate₂, 5mM HEPES, pH 7.5 with NaOH). Voltage protocols from a holding potentialof −80 mV failed to evoke currents different from control oocytes.

[0277] Because the expression pattern of rSK2 is similar to that ofmGluRla, a metabotropic glutamate receptor (Houamed et al., Science,252:1318-1321 (1991); Masu et al., Nature, 349:760-765 (1991)), mGluRlamRNA was injected with or without the SK mRNAS. Addition of glutamate (1mM) to the bath comprising the oocyte injected with mGluR1a mRNA aloneevoked a transient inward current due to activation of endogenouscalcium-activated chloride channels following the release ofintracellular calcium (Houamed et al., Science, 252:1318-1321 (1991);Masu et al., Nature, 349:760-765 (1991)). Similar results were obtainedin six other oocytes injected with mGluR1a. Voltage ramps from −120 to60 mV applied near the peak of the inward response evoked an outwardlyrectifying current that reversed at −25 mv, near the Cl⁻ reversalpotential. Addition of glutamate (1 mM) to oocytes coinjected withmGluR1a and rSK2 mRNA evoked the transient calcium-activated chloridecurrent observed with mGluR1a injected oocytes, followed by a largetransient outward current. Similar results were obtained in 14 otheroocytes coinjected with mGluRla and rSK2. Voltage ramps from −120 to 60mV applied near the peak of the outward response evoked a large inwardlyrectifying current that reversed near −95 mV, close to the K⁺ reversalpotential. This result was obtained with each of the cloned subunits andsuggested that the cloned sequences encode potassium channels.

[0278] Following establishment of the 2-electrode voltage clamp, theoocyte was impaled with a third electrode containing 200 mM EGTA, pHadjusted to 7.2 with KOH. The input resistance was monitored duringimpalement to insure oocyte viability. At the indicated time, 50 nl ofthe EGTA solution was injected into the oocyte. Assuming an oocytevolume of 1 μl, the predicted final concentration of EGTA was 10 mM.Intracellular injection of EGTA abolished both current responses evokedby subsequent application of glutamate indicating that both componentsare calcium-activated. Similar results were obtained in 3 other oocytescoinjected with mGluR1a and rSK2. Current-voltage relation of oocytesinjected with rSK2 mRNA in Cl⁻-free external solution containing 2, 6 or20 mM K⁺. The current was activated by injection of CaCl₂ to a finalconcentration of ˜1 mM (Adelman et al., Neuron, 9:209-216 (1992)).Background current was determined by application of 100 nM apamin. Theapamin-insensitive background current did not vary with external K⁺.

[0279] Two days after injection, the oocytes were soaked for >24 hoursin Cl⁻ free ND96 solution to minimize Cl⁻ currents. In the 2-electroderecording mode, the channel was activated by injection of 5 nl of 200 mMCaCl₂ through a third electrode resulting in a final intracellularconcentration of ˜1 mM Ca²⁺. This procedure resulted in a longer lastingactivation of the K⁺ current than that activated by glutamate in oocytescoinjected with mGluR1a and rSK2. In these oocytes, the reversalpotential was determined relative to background current in 100 nMapamin. The mean reversal potential±S.D. plotted versus [K⁺]₀ yields aslope of 55.4 mV/decade change in [K⁺]₀ and a y-intercept of −110 mV at1 mM [K⁺]₀.

[0280] Macroscopic currents were also recorded from excised patches.Currents were elicited by 2.5 second voltage ramps from −100 to 100 mVin an excised inside-out patch from an oocyte expressing rSK2. Withoutbath applied calcium, currents were not different from control oocytes.Oocytes were injected as described for two-electrode voltage clamprecordings.

[0281] Two to nine days after injection, inside-out macropatces wereexcised into a bath solution containing 116 mM Kgluconate, 4 mM KCl, 10mM HEPES (pH 7.25, adjusted with KOH) supplemented with CaCl₂ and/orEGTA. To obtain nominally Ca-free solution, 1 mM EGTA was added.Alternatively, CaCl₂ was added to the bath solution to give free calciumconcentrations of 1-10 μM. In this case, the proportion of calciumbinding to gluconate was determined by a computer-program (CaBuf)assuming a stability constani for Ca²⁺ gluconate of 15.9 M⁻¹ (Dawson etal., Data for Biochemical Research (Oxford University Press, New York,(1969)). To obtain Ca²+concentrations below 1 μM, 5 mM EGTA was added tothe bath solution and CaCl₂ was added as calculated using the CaBufprogram and published stability constants (Fabiato et al., J. Physiol.,75:463-505 (1979)). For experiments in which Mg²⁺ was added to the bathsolution, MgCl₂ was added to the total concentrations stated in thetext. Under these conditions, binding of Mg²⁺ to gluconate is negligible(stability constant 1.7 M⁻¹).

[0282] Electrodes were pulled from thin-walled, filamented borosilicateglass (World Precision Instruments) and filled with 116 mM Kgluconate, 4mM KCl, 10 mM HEPES (pH 7.25). Electrode resistance was typically 2-5MΩ. Membrane patches were voltage clamped using an Axopatch 200Aamplifier (Axon Instruments). The data were low-pass Bessel filtered at2 kH and acquired using Pulse software (HEKA Electronik). Analysis wasperformed using Pulse, Kaleidograph (Abelbeck), or IGOR (Wavemetrics)software. All experiments were performed at room temperature from aholding potential of −80 mV. 2.5 second voltage ramps from −100 to 100mV were acquired at a sampling frequency of 500 Hz. Alternatively,current-voltage relationships were obtained from the mean current during500 ms commands to voltages between −100 and 100 mV in 20 mV increments,sampled at 5 kHz.

[0283] Addition of 5 μM Ca²⁺ to the intracellular (bath) solution evokeda substantial current. Voltage ramps in symmetrical 120 mM K⁺ and in theabsence of internal Mg²⁺ revealed a current-voltage relationship withslight inward rectification. Voltage steps between −100 and 100 mV, froma holding potential of −80 mV, evoked time-independent currents. Thederived I-V relationship reflects the inward rectification apparent fromvoltage ramps. The current was evoked by voltage steps from aninside-out macropatch excised from an oocyte expressing rSK2. With 5 μMCa²⁺ in the bath, the membrane was stepped from a holding potential of−80 mV to test potentials between −100 and 100 mV and then repolarizedto −50 mV. Currents activated instantaneously and showed no inactivationduring the 500 ms test pulses. Similar results were obtained for hSK1,except that the inward rectification was not as pronounced. Theseresults identify this new family as calcium-activated potassiumchannels.

[0284] 3B. Example 3B describes the electrophysiology of the hIK1channel. All hIK1 channel subunits were subcloned into the oocyteexpression vector pBF (unpublished, graciously provided by Dr. B.Fakler) which provides 5′ and 3′ untranslated regions from the Xenopusβ-globin gene flanking a polylinker containing multiple restrictionsites. In vitro mRNAs were generated using SP6 polymerase (GibcoBRL);following synthesis, mRNAs were evaluated spectrophotometrically and byethidium bromide staining after agarose gel electrophoresis.

[0285] As described above, Xenopus care and handling were in accordancewith the highest standards of institutional guidelines. Frogs underwentno more than two surgeries, separated by at least three weeks, and Allsurgeries were performed using well established techniques. Frogs wereanesthetized with an aerated solution of 3-aminobenzoic acid ethylester. Oocytes were studied 2-14 days after injection with 0.5-5 ng ofmRNA.

[0286] Inside-out macropatches were excised into an intracellularsolution containing 116 mM K-gluconate, 4 mM KCl, 10 mM HEPES (pH 7.2,adjusted with KOH) supplemented with CaCl₂ to give free calciumconcentration of 5 μM; the proportion of calcium binding to gluconatewas determined by a computer program (CaBuf) assuming a stabilityconstant for Ca²⁺ gluconate of 15.9M⁻¹ (Dawson et al., 1969). To obtainCa²+concentrations below 1 μM, 1 mM EGTA was added to the bath solutionand CaCl₂ was added as calculated using the CaBuf program and publishedstability constants (Fabiato and Fabiato, 1979). Electrodes were pulledfrom thin-walled, filamented borosilicate glass (World PrecisionInstruments) and filled with 116 mM K-gluconate, 4 mM KCl, 10 mM HEPES(pH 7.2). Electrode resistance was typically 2-5 MΩ. For outside-outmacropatches, the solutions were reversed. Membrane patches were voltageclamped using an Axopatch 200A amplifier (Axon Instruments). The datawere low-pass Bessel filtered at 1 kH and acquired using Pulse software(HEKA Electronik). Analysis was performed using Pulse, Kaleidagraph(Abelbeck), or IGOR (Wavemetrics) software. Unless otherwise stated allexperiments were performed at room temperature from a holding potentialof 0 mV. 2.5 second voltage ramps from −100 to either 60 or 100 mV wereacquired at a sampling frequency of 500 Hz. Values were expressed asmean±SD. Statistical differences were determined using an unpairedt-test; p values <0.05 were considered significant.

[0287] For single channel recordings, oocytes were bathed in 116 mMKgluconate, 4 mM KCl, 10 mM HEPES, 5 mM EGTA, pH 7.2 adjusted with CaCl₂to yield the reported concentration of free Ca²⁺. All recordings wereperformed in the inside-out patch configuration using thick-walledquartz electrodes (13-15 MΩ) containing 116 mM Kgluconate, 4 mM KCl, 10mM HEPES, pH 7.2. Membrane patches were voltage-clamped with an Axopatch200 amplifier (Axon Instruments). Continuous recordings were low-passBessel filtered at 1 kHz, acquired at 10 kHz using Pulse software (HekaElectronik) and stored directly on a Macintosh Quadra 650. Singlechannel recordings were analyzed with MacTac (SKALAR Instruments) usingthe “50% threshold” technique to estimate event amplitudes and duration,and each transition was visually inspected before being accepted.Amplitude histograms were constructed using MacTacfit (SKALARInstruments). Only events lasting at least 1 ms were included, andamplitude histograms were fitted by single Gaussian distributions. Allexperiments were performed at room temperature.

[0288] The expression of the hIK1 in Xenopus oocytes was readilydetectable. Voltage ramp commands delivered to inside-out patchesexcised into 5 μM Ca²⁺ evoked robust, inwardly rectifying macroscopiccurrent responses, not present in patches from uninjected oocytes (notshown) or inside-out patches bathed in Ca²⁺-free media. Voltage stepcommands evoked large time-independent currents only when Ca²⁺ wasincluded in the (bath) internal solution. Altering the external K⁺concentration (substituted by Na) shifted the reversal potential inaccord with the Nernst prediction for a K⁺-selective conductance (57mV/10-fold change in K⁺). Similar to SK2 channels, currents evoked byvoltage ramp commands were dependent upon the concentration of Ca²⁺applied to the internal face of the membrane.

EXAMPLE 4

[0289] Example 4 describes the calcium sensitivity of rSK2 and hSK1channels.

[0290] Using inside-out micropatches as described above, rSK2 currentsevoked by voltage ramps were shown to dependent upon the concentrationof calcium in the internal (bath) solution. The slope conductance at thereversal potential was plotted as a function of calcium concentrationand the data points fit with the Hill equation. From 8 patches, theaverage K_(d) for calcium was 0.63±0.23 μM. The steep dependence uponcalcium seen from the plot is reflected by a Hill coefficient of4.81±1.46, suggesting that at least two calcium ions are involved inchannel gating. Similar experiments performed with hSK1 yielded a K_(d)of 0.70±0.06 μM and a Hill coefficient of 3.90±0.45.

[0291] To compare hIK1 and SK2, normalized current was plotted as afunction of Ca²⁺ concentration, and the data points fitted with the Hillequation. Both channels showed the same K₀₅ (concentration forhalf-maximal activation, 0.32±0.03 μM (n=7) for hIK1 and 0.31'0.05 μM(n=4) for SK2; p=0.68), but differed in the steepness of theCa²⁺-dependence; SK2 had a Hill coefficient of 3.5±04 (n=4), while hIK1had a Hill coefficient of 1.7±0.3 (n−7, p<0.001). These resultsdemonstrate that bIK1 is also a calcium-activated potassium channel.

EXAMPLE 5

[0292] Example 5 describes the magnesium induced inward rectificationfor the rSK2 channel.

[0293] The inward rectification for rSK2, described above, was observedin the absence of internal cations other than potassium and calcium (5μAM). Native SK channels exhibit inward rectification induced byinternal Mg²⁺ ions (Lancaster et al., J. Neurosci., 11:23-30 (1991)). Inthe hippocampus, SK channels exhibit significant inward rectification inthe presence of internal Mg²⁺ 0 (Id.). Currents were elicited from aninside-out macropatch excised from an oocyte expressing rSK2 in thepresence of the varying concentrations of internal Mg²⁺ and 10 μM Ca²⁺.When different concentrations of Mg²⁺ (0.1-3 mM) were added to thesolution bathing inside-out patches, outward currents were significantlyreduced.

[0294] The concentration- and voltage-dependence of Mg²⁺ induced inwardrectification was examined. A slight decrease of the inward current withincreasing Mg²⁺ was observed. Therefore, the ratio of the outwardcurrent at potentials between 20 and 100 mV to the inward current at−100 mV was plotted as a function of the different concentrations ofinternal Mg²⁺. From multiple experiments, the data points obtained atdifferent Mg²⁺ concentrations and voltages were fit with the Hillequation, yielding an average Hill coefficient of 0.94±0.27 (n=24).Subsequently, the Hill coefficient was fixed at 1, and the mean K_(d)was plotted as a function of the test potential. The K_(d) decreasedwith increasing voltages suggesting that Mg²⁺ block wasvoltage-dependent. K_(d) for Mg²⁺ was obtained from 5 patches as shownin panel B at 20, 40, 60, 80 and 100 mV. Values at each potential wereaveraged, plotted as a function of voltage and fit with the Woodhullequation, K_(d)(0 mV) exp(δzFE/RT) where the K_(d)(0 mV)=6 mM, δ is thefraction of the electric field sensed by the Mg²⁺ ion, 0.30, z is thevalence, 2, and F, E, R, and T have their usual meanings (Woodhull, J.Gen. Physiol., 61:687-708 (1973)). Applying the Woodhull equationsuggested that the Mg2+ ion senses approximately 0.30 of the membraneelectric field.

EXAMPLE 6

[0295] Example 6 describes single channel recordings from oocytes.

[0296] 6A. Example 6A describes single channels were examined usinginside-out patches excised from oocytes expressing rSK2. Addition ofcalcium at submicromolar concentrations induced channel activitynot-seen in controls. A representatives patch showed that 0.2 μM calciumapplied to the bath solution induced openings to a single amplitude.Channel activity increased as the calcium concentration was raised, suchthat in 0.6 μM calcium unitary openings could no longer be resolved.Upon washout of calcium, channel activity disappeared. Channel activityin the presence of 0.4 μM calcium was recorded at several voltages.Similar to macroscopic ramp recordings, channel open probability was notobviously dependent upon voltage.

[0297] Unitary openings measured at several voltages were used toconstruct a single channel I-V relationship. Solutions used were thesame as for macropatch recordings (Example 5). Electrodes were pulledfrom Coming 7052 glass (Gamer) and had resistances of 9-13 MΩ. Data werefiltered at 1 kHz (Bessel), acquired at 10 kHz using Pulse (HEKAElectronik) and stored directly on a Macintosh Quadra 650. Singlechannels were analyzed using MacTac (SKALAR Instruments). The “50%threshold” technique was used to estimate event amplitudes. Thethreshold was adjusted for each opening and each transition wasinspected visually before being accepted. Amplitude histograms wereconstructed using MacTacfit (SKALAR Instruments) and best fit by asingle Gaussian distribution. Channel open probability was estimated asNP(o), the product of the open probability multiplied by the number ofchannels. NP(o) was calculated as the sum of the (dwell time×levelnumber) divided by the total time. N was estimated as the number ofsimultaneously open channels at 0.4 μM calcium. Linear regressionanalysis on three patches from an oocyte expressing either rSK2 or hSK1yielded a mean single channel conductance of 9.9±0.9 pS and 9.2±0.3 pS,respectively.

[0298] 6B. Example 6B describes the single channel conductance of hIK1.The methodology is described above in Example 3B. Stationary recordingsfrom inside-out patches excised into a bathing solution containing0.2-1.0 μM free calcium showed short-duration openings not seen in theabsence of calcium. Representative traces were recorded at −60 mV. Thedegree of channel activity depended upon the concentration of internalcalcium. Reducing intracellular calcium reduced channel activity, andremoving internal calcium abolished channel activity, which returnedafter reapplication of Ca²⁺. Sustained channel activity was seen atmembrane voltages ranging from −100 mV to +100 mV and open probabilitywas not obviously voltage-dependent. For select patchs, the amplitudesof openings were measured, assembled into histograms, and fit byGaussian distributions. The resulting mean amplitudes were used toconstruct the current-voltage relationship. The single channelcurrent-voltage relationship shows inward rectification similar to themacroscopic current-voltage relationship. For this patch, linearregression analysis of the inward current-voltage relationship yielded asingle channel conductance of 35 pS; results from four patches gave aunit conductance of 38±4 pS. Measurements of the outward conductancewere more variable, ranging from 5 to 12 pS.

EXAMPLE 7

[0299] Example 7 describes the pharmacology of the novel rat and humanpotassium channels.

[0300] 7A. Macroscopic rSK2 currents were recorded in 5 μM Ca²⁺ frominside-out macropatches with either 0 or 60 pM apamin or 0 or 2 μMd-tubocurare in the patch pipette described in Example 3. The functionalcharacteristics of the cloned channels are reminiscent of the SK classof calcium-activated potassium channels described in neurons (Lancasterand Adams, J. Neurophysiol., 55:1268-1282 (1986); Lancasteretal., J.Neurosci., 11:23-30 (1991); Sah et al., J. Neurophysiol., 68:1834-1841(1992)), skeletal muscle (Blatz and Magleby, Nature, 323:718-720(1986)), adrenal chromaffin cells (Park, J. Physiol., 481:555-570((1994); Artalejo et al., Pflugers Archiv., 423:97-103 (1993)), andT-lymphocytes (Grissmer et al., J. Gen. Physiol., 99:63-84 (1992)).Native SK channels present a distinct pharmacology. They are not blockedby the scorpion peptide, charybdotoxin (CTX), a potent blocker of BKpotassium channels (Miller et al., Nature, 313:316-318 (1985)). However,many but not all SK channels are blocked by the bee venom toxin, apamin,and the plant alkyloid, d-tubocurare (dTC; Zhang and McBain, J.Physiol., 488:661-672 (1995), Park, J. Physiol., 481:555-570 (1994); Dunet al., J. Physiol., 375:499-514 (1986)). Application of 500 nM CTX didnot block rSK2 or hSK1, but abolished the activity of hSlo BK currents.rSK2 currents were potently blocked by picomolar concentrations ofapamin with a K_(d) of 63 pM. In contrast, application of 100 nM apamindid not affect hSK-1 currents (n=8). dTC also blocked rSK2 currents witha K_(d) of 2.4 μM, while hSK1 was approximately 30-fold less sensitive,with a K_(d) of 76.2 μM.

[0301] 7B. For the pharmacology tests of hIK1, Clotrimazole was fromSigma, ketoconazole and iberiotoxin were from RPI, apamin was fromCalbiochem, charybdotoxin was the generous gift of Dr. Chris Miller. Thefunctional characteristics of hIK1 are remeniscent of intermediateconductance calcium-activated K⁺ channels described from red blood cells(the Gardos channel; Gardos, 1958) and other tissues. Native IK channelspresent a distinguishing pharmacology; being-blocked by charybdotoxin(CTX) but, different from large conductance voltage- and Ca²⁺-activatedK⁺ channels (BK channels), are not blocked by iberiotoxin. Also, IKchannels are not sensitive to the bee venom peptide toxin apamin, ablocker of certain native and cloned SK channels. In addition, some IKchannels, notably the Gardos channel, are sensitive to several imidazolederivatives such as clotrimazole, but are not sensitive to others suchas ketoconazole. hIK1 currents were potently blocked by CTX, with a K₁of 2.5 nM (n=4), while 50 nM IBX blocked only 15±3%. Human IK1 wassensitive to clotrimazole with a Ki of 24.8 nM, but was only 24±6%blocked by 10 μM ketoconazole. 100 nM apamin reduced hIK1 currents byonly 12±5%.

[0302] All publications and patents mentioned in this specification areherein incorporated by reference into the specification to the sameextent as if each individual publication or patent was specifically andindividually indicated to be incorporated herein by reference.

1 48 561 amino acids amino acid <Unknown> linear protein Protein 1..561/note= “human small conductance, calcium-activated potassium channelprotein 1 (hSK1)” 1 Met Pro Gly Pro Arg Ala Ala Cys Ser Glu Pro Asn ProCys Thr Gln 1 5 10 15 Val Val Met Asn Ser His Ser Tyr Asn Gly Ser ValGly Arg Pro Leu 20 25 30 Gly Ser Gly Pro Gly Ala Leu Gly Arg Asp Pro ProAsp Pro Glu Ala 35 40 45 Gly His Pro Pro Gln Pro Pro His Ser Pro Gly LeuGln Val Val Val 50 55 60 Ala Lys Ser Glu Pro Ala Arg Pro Ser Pro Gly SerPro Arg Gly Gln 65 70 75 80 Pro Gln Asp Gln Asp Asp Asp Glu Asp Asp GluGlu Asp Glu Ala Gly 85 90 95 Arg Gln Arg Ala Ser Gly Lys Pro Ser Asn ValGly His Arg Leu Gly 100 105 110 His Arg Arg Ala Leu Phe Glu Lys Arg LysArg Leu Ser Asp Tyr Ala 115 120 125 Leu Ile Phe Gly Met Phe Gly Ile ValVal Met Val Thr Glu Thr Glu 130 135 140 Leu Ser Trp Gly Val Tyr Thr LysGlu Ser Leu Tyr Ser Phe Ala Leu 145 150 155 160 Lys Cys Leu Ile Ser LeuSer Thr Ala Ile Leu Leu Gly Leu Val Val 165 170 175 Leu Tyr His Ala ArgGlu Ile Gln Leu Phe Met Val Asp Asn Gly Ala 180 185 190 Asp Asp Trp ArgIle Ala Met Thr Cys Glu Arg Val Phe Leu Ile Ser 195 200 205 Leu Glu LeuAla Val Cys Ala Ile His Pro Val Pro Gly His Tyr Arg 210 215 220 Phe ThrTrp Thr Ala Arg Leu Ala Phe Thr Tyr Ala Pro Ser Val Ala 225 230 235 240Glu Ala Asp Val Asp Val Leu Leu Ser Ile Pro Met Phe Leu Arg Leu 245 250255 Tyr Leu Leu Gly Arg Val Met Leu Leu His Ser Lys Ile Phe Thr Asp 260265 270 Ala Ser Ser Arg Ser Ile Gly Ala Leu Asn Lys Ile Thr Phe Asn Thr275 280 285 Arg Phe Val Met Lys Thr Leu Met Thr Ile Cys Pro Gly Thr ValLeu 290 295 300 Leu Val Phe Ser Ile Ser Ser Trp Ile Ile Ala Ala Trp ThrVal Arg 305 310 315 320 Val Cys Glu Arg Tyr His Asp Lys Gln Glu Val ThrSer Asn Phe Leu 325 330 335 Gly Ala Met Trp Leu Ile Ser Ile Thr Phe LeuSer Ile Gly Tyr Gly 340 345 350 Asp Met Val Pro His Thr Tyr Cys Gly LysGly Val Cys Leu Leu Thr 355 360 365 Gly Ile Met Gly Ala Gly Cys Thr AlaLeu Val Val Ala Val Val Ala 370 375 380 Arg Lys Leu Glu Leu Thr Lys AlaGlu Lys His Val His Asn Phe Met 385 390 395 400 Met Asp Thr Gln Leu ThrLys Arg Val Lys Asn Ala Ala Ala Asn Val 405 410 415 Leu Arg Glu Thr TrpLeu Ile Tyr Lys His Thr Arg Leu Val Lys Lys 420 425 430 Pro Asp Gln AlaArg Val Arg Lys His Gln Arg Lys Phe Leu Gln Ala 435 440 445 Ile His GlnAla Gln Lys Leu Arg Ser Val Lys Ile Glu Gln Gly Lys 450 455 460 Leu AsnAsp Gln Ala Asn Thr Leu Thr Asp Leu Ala Lys Thr Gln Thr 465 470 475 480Val Met Tyr Asp Leu Val Ser Glu Leu His Ala Gln His Glu Glu Leu 485 490495 Glu Ala Arg Leu Ala Thr Leu Glu Ser Arg Leu Asp Ala Leu Gly Ala 500505 510 Ser Leu Gln Ala Leu Pro Gly Leu Ile Ala Gln Ala Ile Arg Pro Pro515 520 525 Pro Pro Pro Leu Pro Pro Arg Pro Gly Pro Gly Pro Gln Asp GlnAla 530 535 540 Ala Arg Ser Ser Pro Cys Arg Trp Thr Pro Val Ala Pro SerAsp Cys 545 550 555 560 Gly 580 amino acids amino acid <Unknown> linearprotein Protein 1..580 /note= “rat small conductance, calcium-activatedpotassium channel protein 2 (rSK2)” Region 135..462 /note= “core regionof rSK2” 2 Met Ser Ser Cys Arg Tyr Asn Gly Gly Val Met Arg Pro Leu SerAsn 1 5 10 15 Leu Ser Ser Ser Arg Arg Asn Leu His Glu Met Asp Ser GluAla Gln 20 25 30 Pro Leu Gln Pro Pro Ala Ser Val Val Gly Gly Gly Gly GlyAla Ser 35 40 45 Ser Pro Ser Ala Ala Ala Ala Ala Ser Ser Ser Ala Pro GluIle Val 50 55 60 Val Ser Lys Pro Glu His Asn Asn Ser Asn Asn Leu Ala LeuTyr Gly 65 70 75 80 Thr Gly Gly Gly Gly Ser Thr Gly Gly Gly Gly Gly GlyGly Gly Gly 85 90 95 Gly Gly Gly Ser Gly His Gly Ser Ser Ser Gly Thr LysSer Ser Lys 100 105 110 Lys Lys Asn Gln Asn Ile Gly Tyr Lys Leu Gly HisArg Arg Ala Leu 115 120 125 Phe Glu Lys Arg Lys Arg Leu Ser Asp Tyr AlaLeu Ile Phe Gly Met 130 135 140 Phe Gly Ile Val Val Met Val Ile Glu ThrGlu Leu Ser Trp Gly Ala 145 150 155 160 Tyr Asp Lys Ala Ser Leu Tyr SerLeu Ala Leu Lys Cys Leu Ile Ser 165 170 175 Leu Ser Thr Ile Ile Leu LeuGly Leu Ile Ile Val Tyr His Ala Arg 180 185 190 Glu Ile Gln Leu Phe MetVal Asp Asn Gly Ala Asp Asp Trp Arg Ile 195 200 205 Ala Met Thr Tyr GluArg Ile Phe Phe Ile Cys Leu Glu Ile Leu Val 210 215 220 Cys Ala Ile HisPro Ile Pro Gly Asn Tyr Thr Phe Thr Trp Thr Ala 225 230 235 240 Arg LeuAla Phe Ser Tyr Ala Pro Ser Thr Thr Thr Ala Asp Val Asp 245 250 255 IleIle Leu Ser Ile Pro Met Phe Leu Arg Leu Tyr Leu Ile Ala Arg 260 265 270Val Met Leu Leu His Ser Lys Leu Phe Thr Asp Ala Ser Ser Arg Ser 275 280285 Ile Gly Ala Leu Asn Lys Ile Asn Phe Asn Thr Arg Phe Val Met Lys 290295 300 Thr Leu Met Thr Ile Cys Pro Gly Thr Val Leu Leu Val Phe Ser Ile305 310 315 320 Ser Leu Trp Ile Ile Ala Ala Trp Thr Val Arg Ala Cys GluArg Tyr 325 330 335 His Asp Gln Gln Asp Val Thr Ser Asn Phe Leu Gly AlaMet Trp Leu 340 345 350 Ile Ser Ile Thr Phe Leu Ser Ile Gly Tyr Gly AspMet Val Pro Asn 355 360 365 Thr Tyr Cys Gly Lys Gly Val Cys Leu Leu ThrGly Ile Met Gly Ala 370 375 380 Gly Cys Thr Ala Leu Val Val Ala Val ValAla Arg Lys Leu Glu Leu 385 390 395 400 Thr Lys Ala Glu Lys His Val HisAsn Phe Met Met Asp Thr Gln Leu 405 410 415 Thr Lys Arg Val Lys Asn AlaAla Ala Asn Val Leu Arg Glu Thr Trp 420 425 430 Leu Ile Tyr Lys Asn ThrLys Leu Val Lys Lys Ile Asp His Ala Lys 435 440 445 Val Arg Lys His GlnArg Lys Phe Leu Gln Ala Ile His Gln Leu Arg 450 455 460 Ser Val Lys MetGlu Gln Arg Lys Leu Asn Asp Gln Ala Asn Thr Leu 465 470 475 480 Val AspLeu Ala Lys Thr Gln Asn Ile Met Tyr Asp Met Ile Ser Asp 485 490 495 LeuAsn Glu Arg Ser Glu Asp Phe Glu Lys Arg Ile Val Thr Leu Glu 500 505 510Thr Lys Leu Glu Thr Leu Ile Gly Ser Ile His Ala Leu Pro Gly Leu 515 520525 Ile Ser Gln Thr Ile Arg Gln Gln Gln Arg Asp Phe Ile Glu Thr Gln 530535 540 Met Glu Asn Tyr Asp Lys His Val Thr Tyr Asn Ala Glu Arg Ser Arg545 550 555 560 Ser Ser Ser Arg Arg Arg Arg Ser Ser Ser Thr Ala Pro ProThr Ser 565 570 575 Ser Glu Ser Ser 580 553 amino acids amino acid<Unknown> linear protein Protein 1..553 /note= “N-terminally truncatedform of rat small conductance, calcium-activated potassium channelprotein 3 (rSK3)” Region 109..436 /note= “core region of rSK3” 3 Met SerSer Cys Lys Tyr Ser Gly Gly Val Met Lys Pro Leu Ser Arg 1 5 10 15 LeuSer Ala Ser Arg Arg Asn Leu Ile Glu Ala Glu Pro Glu Gly Gln 20 25 30 ProLeu Gln Leu Phe Ser Pro Ser Asn Pro Pro Glu Ile Ile Ile Ser 35 40 45 SerArg Glu Asp Asn His Ala His Gln Thr Leu Leu His His Pro Asn 50 55 60 AlaThr His Asn His Gln His Ala Gly Thr Thr Ala Gly Ser Thr Thr 65 70 75 80Phe Pro Lys Ala Asn Lys Arg Lys Asn Gln Asn Ile Gly Tyr Lys Leu 85 90 95Gly His Arg Arg Ala Leu Phe Glu Lys Arg Lys Arg Leu Ser Asp Tyr 100 105110 Ala Leu Ile Phe Gly Met Phe Gly Ile Val Val Met Val Ile Glu Thr 115120 125 Glu Leu Ser Trp Gly Leu Tyr Ser Lys Asp Ser Met Phe Ser Leu Ala130 135 140 Leu Lys Cys Leu Ile Ser Leu Ser Thr Ile Ile Leu Leu Gly LeuIle 145 150 155 160 Ile Ala Tyr His Thr Arg Glu Val Gln Leu Phe Val IleAsp Asn Gly 165 170 175 Ala Asp Asp Trp Arg Ile Ala Met Thr Tyr Glu ArgIle Leu Tyr Ile 180 185 190 Ser Leu Glu Met Leu Val Cys Ala Ile His ProIle Pro Gly Glu Tyr 195 200 205 Lys Phe Phe Trp Thr Ala Arg Leu Ala PheSer Tyr Thr Pro Ser Arg 210 215 220 Ala Glu Ala Asp Val Asp Ile Ile LeuSer Ile Pro Met Phe Leu Arg 225 230 235 240 Leu Tyr Leu Ile Ala Arg ValMet Leu Leu His Ser Lys Leu Phe Thr 245 250 255 Asp Ala Ser Ser Arg SerIle Gly Ala Leu Asn Lys Ile Asn Phe Asn 260 265 270 Thr Arg Phe Val MetLys Thr Leu Met Thr Ile Cys Pro Gly Thr Val 275 280 285 Leu Leu Met PheSer Ile Ser Leu Trp Ile Ile Ala Ala Trp Thr Val 290 295 300 Arg Val CysGlu Arg Tyr His Asp Gln Gln Asp Val Thr Ser Asn Phe 305 310 315 320 LeuGly Ala Met Trp Leu Ile Ser Ile Thr Phe Leu Ser Ile Gly Tyr 325 330 335Gly Asp Met Val Pro His Thr Tyr Cys Gly Lys Gly Val Cys Leu Leu 340 345350 Thr Gly Ile Met Gly Ala Gly Cys Thr Ala Leu Val Val Ala Val Val 355360 365 Ala Arg Lys Leu Glu Leu Thr Lys Ala Glu Lys His Val His Asn Phe370 375 380 Met Met Asp Thr Gln Leu Thr Lys Arg Ile Lys Asn Ala Ala AlaAsn 385 390 395 400 Val Leu Arg Glu Thr Trp Leu Ile Tyr Lys His Thr LysLeu Leu Lys 405 410 415 Lys Ile Asp His Ala Lys Val Arg Lys His Gln ArgLys Phe Leu Gln 420 425 430 Ala Ile His Gln Leu Arg Gly Val Lys Met GluGln Arg Lys Leu Ser 435 440 445 Asp Gln Ala Asn Thr Leu Val Asp Leu SerLys Met Gln Asn Val Met 450 455 460 Tyr Asp Leu Ile Thr Glu Leu Asn AspArg Ser Glu Asp Leu Glu Lys 465 470 475 480 Gln Ile Gly Ser Leu Glu SerLys Leu Glu His Leu Thr Ala Ser Phe 485 490 495 Asn Ser Leu Pro Leu LeuIle Ala Asp Thr Leu Arg Gln Gln Gln Gln 500 505 510 Gln Leu Leu Thr AlaPhe Val Glu Ala Arg Gly Ile Ser Val Ala Val 515 520 525 Gly Thr Ser HisAla Pro Pro Ser Asp Ser Pro Ile Gly Ile Ser Ser 530 535 540 Thr Ser PhePro Glu Phe Leu Ile Phe 545 550 458 amino acids amino acid <Unknown>linear protein Protein 1..458 /note= “rat small conductance,calcium-activated potassium channel protein 1 (rSK1)” 4 Ser Gly Lys ProPro Thr Val Ser His Arg Leu Gly His Arg Arg Ala 1 5 10 15 Leu Phe GluLys Arg Lys Arg Leu Ser Asp Tyr Ala Leu Ile Phe Gly 20 25 30 Met Phe GlyIle Val Val Met Val Thr Glu Thr Glu Leu Ser Trp Gly 35 40 45 Val Tyr ThrLys Glu Ser Leu Cys Ser Phe Ala Leu Lys Cys Leu Ile 50 55 60 Ser Leu SerThr Val Ile Leu Leu Gly Leu Val Ile Leu Tyr His Ala 65 70 75 80 Arg GluIle Gln Leu Phe Leu Val Asp Asn Gly Ala Asp Asp Trp Arg 85 90 95 Ile AlaMet Thr Trp Glu Arg Val Ser Leu Ile Ser Leu Glu Leu Ala 100 105 110 ValCys Ala Ile His Pro Val Pro Gly His Tyr Arg Phe Thr Trp Thr 115 120 125Ala Arg Leu Ala Phe Ser Leu Val Pro Ser Ala Ala Glu Ala Asp Val 130 135140 Asp Val Leu Leu Ser Ile Pro Met Phe Leu Arg Leu Tyr Leu Leu Ala 145150 155 160 Arg Val Met Leu Leu His Ser Arg Ile Phe Thr Asp Ala Ser SerArg 165 170 175 Ser Ile Gly Ala Leu Asn Arg Val Thr Phe Asn Thr Arg PheVal Thr 180 185 190 Lys Thr Leu Met Thr Ile Cys Pro Gly Thr Val Leu LeuVal Phe Ser 195 200 205 Ile Ser Ser Trp Ile Val Ala Ala Trp Thr Val ArgVal Cys Glu Arg 210 215 220 Tyr His Asp Lys Gln Glu Val Thr Ser Asn PheLeu Gly Ala Met Trp 225 230 235 240 Leu Ile Ser Ile Thr Phe Leu Ser IleGly Tyr Gly Asp Met Val Pro 245 250 255 His Thr Tyr Cys Gly Lys Gly ValCys Leu Leu Thr Gly Ile Met Gly 260 265 270 Ala Gly Cys Thr Ala Leu ValVal Ala Val Val Ala Arg Lys Leu Glu 275 280 285 Leu Thr Lys Ala Glu LysHis Val His Asn Phe Met Met Asp Thr Gln 290 295 300 Leu Thr Lys Arg ValLys Asn Ala Ala Ala Asn Val Leu Arg Glu Thr 305 310 315 320 Trp Leu IleTyr Lys His Thr Arg Leu Val Lys Lys Pro Asp Gln Ser 325 330 335 Arg ValArg Lys His Gln Arg Lys Phe Leu Gln Ala Ile His Gln Ala 340 345 350 GlnLys Leu Arg Thr Val Lys Ile Glu Gln Gly Lys Val Asn Asp Gln 355 360 365Ala Asn Thr Leu Ala Asp Leu Ala Lys Ala Gln Ser Ile Ala Tyr Glu 370 375380 Val Val Ser Glu Leu Gln Ala Gln Gln Glu Glu Leu Glu Ala Arg Leu 385390 395 400 Ala Ala Leu Glu Ser Arg Leu Asp Val Leu Gly Ala Ser Leu GlnAla 405 410 415 Leu Pro Ser Leu Ile Ala Gln Ala Ile Cys Pro Leu Pro ProPro Trp 420 425 430 Pro Gly Pro Ser His Leu Thr Thr Ala Ala Gln Ser ProGln Ser His 435 440 445 Trp Leu Pro Thr Thr Ala Ser Asp Cys Gly 450 45524 base pairs nucleic acid single linear DNA 5 ATGCCGGGTC CCCGGGCGGCCTGC 24 24 base pairs nucleic acid single linear DNA 6 TCACCCGCAGTCCGAGGGGG CCAC 24 24 base pairs nucleic acid single linear DNA 7ATGAGCAGCT GCAGGTACAA CGGG 24 24 base pairs nucleic acid single linearDNA 8 CTAGCTACTC TCAGATGAAG TTGG 24 24 base pairs nucleic acid singlelinear DNA 9 ATGAGCTCCT GCAAATACAG CGGT 24 20 base pairs nucleic acidsingle linear DNA 10 TTAGCAACTG CTTGAACTTG 20 24 base pairs nucleic acidsingle linear DNA 11 TCAGGGAAGC CCCCGACCGT CAGT 24 24 base pairs nucleicacid single linear DNA 12 TCACCCACAG TCTGATGCCG TGGT 24 1683 base pairsnucleic acid single linear cDNA - 1..1683 /note= “human smallconductance, calcium-activated potassium channel protein 1 (hSK1) cDNA”13 ATGCCGGGTC CCCGGGCGGC CTGCAGCGAG CCCAACCCCT GCACCCAGGT AGTCATGAAC 60AGCCACAGCT ACAATGGCAG CGTGGGGCGG CCGCTGGGCA GCGGGCCGGG CGCCCTGGGA 120CGAGACCCTC CGGACCCTGA GGCCGGCCAC CCCCCACAAC CCCCGCACAG CCCGGGCCTC 180CAGGTGGTAG TGGCCAAGAG TGAGCCAGCC CGGCCCTCAC CCGGCAGCCC CCGGGGGCAG 240CCCCAGGACC AGGACGATGA CGAGGATGAT GAGGAAGATG AGGCCGGCAG GCAGAGAGCC 300TCGGGGAAAC CCTCAAATGT GGGCCACCGC CTGGGCCACC GGCGGGCGCT CTTCGAGAAG 360CGGAAGCGCC TCAGCGACTA TGCCCTCATT TTCGGCATGT TTGGCATCGT CGTCATGGTG 420ACGGAGACCG AGCTGTCCTG GGGGGTGTAC ACCAAGGAGT CTCTGTACTC ATTCGCACTC 480AAATGCCTCA TGAGCCTCTC CACGGCCATC CTGCTGGGTC TCGTTGTCCT CTACCATGCC 540CGGGAGATCC AGCTGTTCAT GGTGGACAAC GGGGCTGATG ACTGGCGCAT CGCCATGACC 600TGCGAGCGCG TGTTCCTCAT CTCGCTAGAG CTGGCAGTGT GCGCCATTCA CCCGGTGCCC 660GGCCACTACC GCTTCACGTG GACGGCGCGG CTGGCCTTCA CGTACGCGCC CTCGGTGGCC 720GAGGCCGACG TGGACGTGCT GCTGTCCATC CCCATGTTCC TGCGCCTCTA CCTGCTGGGC 780CGGGTGATGC TACTGCACAG CAAAATCTTC ACGGACGCCT CGAGCCGCAG CATCGGGGCC 840CTCAACAAGA TCACCTTCAA CACGCGCTTC GTCATGAAGA CACTCATGAC CATCTGCCCC 900GGCACCGTGC TGCTGGTCTT CAGCATCTCC TCCTGGATCA TCGCAGCCTG GACCGTGCGC 960GTCTGCGAGA GGTACCACGA CAAGCAGGAA GTGACCAGCA ACTTCCTGGG GGCCATGTGG 1020CTGATTTCCA TCACCTTCCT CTCCATTGGC TACGGCGACA TGGTGCCCCA CACCTACTGC 1080GGGAAGGGTG TGTGCCTGCT CACTGGCATC ATGGGAGCTG GCTGTACCGC GCTCGTGGTG 1140GCTGTGGTGG CTCGGAAGCT GGAGCTCACC AAGGCTGAGA AGCACGTGCA CAACTTCATG 1200ATGGACACTC AGCTCACCAA GCGGGTAAAA AACGCCGCTG CTAACGTTCT CAGGGAGACG 1260TGGCTCATCT ACAAACATAC CAGGCTGGTG AAGAAGCCAG ACCAAGCCCG GGTTCGGAAA 1320CACCAGCGTA AGTTCCTCCA AGCCATCCAT CAGGCTCAGA AGCTCCGGAG TGTGAAGATC 1380GAGCAAGGGA AGCTGAACGA CCAGGCTAAC ACGCTTACCG ACCTAGCCAA GACCCAGACC 1440GTCATGTACG ACCTTGTATC GGAGCTGCAC GCTCAGCACG AGGAGCTGGA GGCCCGCCTG 1500GCCACCCTGG AAAGCCGCTT GGATGCGCTG GGTGCCTCTC TACAGGCCCT GCCTGGCCTC 1560ATCGCCCAAG CCATACGCCC ACCCCCGCCT CCCCTGCCTC CCAGGCCCGG CCCCGGCCCC 1620CAAGACCAGG CAGCCCGGAG CTCCCCCTGC CGGTGGACGC CCGTGGCCCC CTCGGACTGC 1680GGG 1683 1374 base pairs nucleic acid single linear cDNA - 1..1374/note= “rat small conductance, calcium-activated potassium channelprotein 1 (rSK1) cDNA” 14 TCAGGGAAGC CCCCGACCGT CAGTCACCGC CTGGGCCACCGTAGGGCCCT CTTCGAGAAG 60 CGTAAACGAC TCAGTGACTA TGCACTCATC TTTGGCATGTTCGGGATTGT CGTCATGGTG 120 ACAGAAACAG AGCTGTCCTG GGGTGTGTAC ACCAAGGAGTCTCTGTGCTC ATTCGCCCTG 180 AAATGCCTAA TCAGCCTCTC CACTGTCATC CTGCTTGGCCTTGTCATCCT CTACCACGCC 240 CGAGAGATCC AGCTGTTCCT GGTGGACAAT GGTGCCGATGACTGGCGCAT TGCCATGACG 300 TGGGAGCGAG TGTCCCTGAT CTCGCTGGAG TTGGCTGTGTGTGCCATCCA CCCAGTGCCT 360 GGCCACTACC GCTTCACATG GACGGCGCGG CTGGCCTTCTCCCTGGTGCC GTCAGCAGCC 420 GAGGCGGATG TGGATGTGCT TCTGTCCATC CCCATGTTTCTGCGCCTCTA TCTGCTGGCT 480 CGGGTCATGC TCCTGCACAG CCGCATCTTC ACGGACGCATCCAGTCGCAG CATCGGAGCC 540 CTGAACCGTG TCACCTTCAA CACACGCTTT GTCACCAAGACACTCATGAC CATCTGCCCT 600 GGCACCGTGC TGTTGGTCTT CAGCATCTCC TCCTGGATCGTCGCTGCATG GACAGTGCGC 660 GTGTGTGAGA GGTACCATGA TAAACAGGAA GTGACCAGCAACTTCCTGGG GGCCATGTGG 720 CTCATCTCCA TTACCTTCCT GTCCATCGGC TACGGGGACATGGTGCCGCA CACCTACTGT 780 GGGAAGGGCG TGTGTCTGCT CACCGGCATC ATGGGAGCAGGCTGCACTGC ACTCGTGGTG 840 GCCGTCGTGG CCCGCAAGTT GGAACTCACC AAGGCTGAGAAACACGTGCA CAACTTCATG 900 ATGGACACAC AGCTCACCAA GCGGGTTAAA AACGCCGCTGCAAACGTTCT CAGGGAGACA 960 TGGCTCATCT ACAAACACAC CAGGCTAGTG AAGAAGCCAGACCAAAGCCG GGTTCGGAAA 1020 CACCAGCGTA AGTTCCTTCA GGCCATCCAT CAGGCGCAGAAGCTCCGGAC TGTGAAGATT 1080 GAACAAGGGA AGGTGAATGA TCAGGCCAAC ACGCTGGCTGACCTGGCCAA GGCACAGAGC 1140 ATCGCATATG AGGTGGTGTC GGAGCTGCAG GCCCAGCAGGAGGAGTTGGA GGCCCGTCTG 1200 GCTGCCCTGG AGAGCCGCCT GGATGTCCTA GGCGCCTCCCTGCAGGCCCT ACCAAGTCTC 1260 ATAGCCCAAG CCATATGCCC TCTACCACCA CCCTGGCCCGGGCCCAGTCA CCTGACCACA 1320 GCCGCCCAGA GCCCACAAAG CCACTGGCTG CCCACCACGGCATCAGACTG TGGG 1374 1740 base pairs nucleic acid single linear cDNA -1..1740 /note= “rat small conductance, calcium-activated potassiumchannel protein 2 (rSK2) cDNA” 15 ATGAGCAGCT GCAGGTACAA CGGGGGCGTCATGCGTCCGC TCAGCAACTT GAGCTCGTCC 60 CGCCGGAACC TGCACGAGAT GGACTCAGAGGCTCAGCCCC TGCAGCCCCC AGCGTCGGTT 120 GTAGGAGGAG GTGGTGGTGC GTCCTCCCCGTCTGCTGCCG CCGCCGCCTC ATCCTCAGCC 180 CCAGAGATCG TGGTGTCTAA GCCGGAGCACAACAATTCTA ACAACCTGGC GCTCTACGGA 240 ACTGGCGGCG GAGGCAGCAC CGGAGGCGGCGGCGGCGGCG GCGGCGGCGG CGGCGGCAGC 300 GGGCATGGCA GCAGCAGCGG CACTAAGTCCAGCAAAAAGA AGAACCAGAA CATCGGCTAT 360 AAGCTGGGCC ATCGGCGTGC CCTGTTTGAGAAGCGCAAGC GGCTCAGCGA CTATGCGCTC 420 ATCTTCGGCA TGTTCGGCAT CGTGGTCATGGTCATCGAGA CCGAGCTGTC GTGGGGCGCC 480 TACGACAAGG CGTCGCTGTA TTCTTTAGCTCTGAAATGCC TTATCAGTCT CTCCACGATC 540 ATCCTGCTTG GTCTGATCAT CGTATACCACGCCAGGGAAA TACAGTTATT CATGGTGGAC 600 AATGGAGCAG ATGACTGGAG AATAGCCATGACTTATGAAC GTATTTTCTT CATCTGCTTG 660 GAAATACTGG TGTGTGCTAT TCATCCCATCCCTGGGAATT ATACGTTCAC ATGGACAGCC 720 CGGCTTGCCT TCTCCTATGC CCCTTCCACAACCACTGCAG ACGTGGATAT TATTTTATCT 780 ATACCAATGT TCTTAAGACT CTATCTGATTGCCAGAGTCA TGCTATTACA TAGCAAACTT 840 TTCACCGATG CCTCCTCTAG AAGCATTGGGGCACTTAATA AGATAAACTT CAATACGCGT 900 TTTGTTATGA AGACTTTAAT GACTATCTGCCCAGGAACTG TGCTCTTGGT TTTTAGTATC 960 TCGTTATGGA TAATTGCCGC ATGGACTGTCCGAGCTTGTG AAAGGTACCA TGATCAACAG 1020 GATGTCACTA GCAACTTCCT TGGAGCAATGTGGTTGATAT CAATAACTTT TCTCTCCATT 1080 GGTTATGGTG ACATGGTACC TAACACATACTGTGGGAAAG GAGTCTGCTT GCTTACCGGA 1140 ATAATGGGTG CAGGTTGCAC AGCCTTGGTGGTAGCCGTAG TGGCAAGGAA GCTAGAACTT 1200 ACCAAAGCAG AAAAGCATGT GCACAATTTCATGATGGATA CTCAGCTGAC CAAAAGAGTA 1260 AAAAACGCAG CCGCCAATGT ACTCAGGGAAACGTGGTTAA TCTACAAAAA CACAAAGCTA 1320 GTGAAAAAGA TCGACCATGC AAAAGTAAGGAAGCATCAAC GGAAATTCTT ACAAGCTATT 1380 CATCAATTAA GAAGTGTGAA GATGGAACAGAGGAAACTGA ATGACCAAGC GAATACGCTA 1440 GTGGATCTGG CAAAGACCCA AGATATCATGTATGATATGA TTTCCGACTT AAATGTAAGG 1500 AGTGAAGACT TTGAGAAAAG GATCGTCACCCTGGAAACAA AATTAGAAAC TTTGATTGGT 1560 AGCATTCATG CCCTCCCTGG GCTTATCAGCCAGACCATCA GACAGCAGCA AAGGGACTTC 1620 ATAGAGACAC AGATGGAGAA CTATGACAAGCATGTCACCT ACAATGCTGA GCGTTCCCGG 1680 TCCTCGTCCA GGAGGCGGCG GTCCTCCTCCACAGCGCCAC CAACTTCATC TGAGAGTAGC 1740 1659 base pairs nucleic acidsingle linear cDNA - 1..1659 /note= “N-terminally truncated cDNA for ratsmall conductance, calcium-activated potassium channel protein 3 (rSK3)”16 ATGAGCTCCT GCAAATACAG CGGTGGGGTC ATGAAGCCCC TCAGCCGCCT CAGCGCCTCT 60CGGAGAAACC TTATCGAGGC CGAGCCTGAG GGCCAACCCC TCCAGCTCTT CAGTCCCAGC 120AACCCCCCAG AGATTATCAT CTCCTCCAGG GAGGATAACC ATGCCCACCA GACTCTGCTC 180CATCACCCCA ACGCTACCCA CAACCACCAG CATGCCGGCA CCACTGCTGG CAGCACCACC 240TTCCCCAAAG CCAACAAGCG GAAAAACCAA AACATTGGCT ATAAGCTGGG GCACAGGAGG 300GCCCTGTTTG AAAAGAGAAA GCGACTGAGT GACTATGCTC TGATTTTTGG GATGTTTGGA 360ATTGTTGTTA TGGTGATAGA GACCGAACTG TCTTGGGGTT TGTACTCAAA GGATTCCATG 420TTTTCGTTGG CCCTGAAATG CCTTATCAGT TTATCCACCA TCATCCTGCT TGGTTTGATC 480ATCGCCTACC ACACAAGGGA AGTACAGCTC TTTGTGATCG ACAATGGTGC AGATGACTGG 540CGGATAGCCA TGACCTATGA GCGCATCCTC TACATCAGCC TGGAGATGCT GGTGTGCGCC 600ATCCACCCCA TTCCTGGAGA GTACAAGTTC TTCTGGACGG CACGCCTGGC CTTCTCCTAC 660ACCCCCTCTC GGGCAGAGGC TGACGTGGAC ATTATTCTGT CCATCCCCAT GTTCTTGCGC 720CTATACCTGA TCGCCCGAGT CATGCTGCTA CATAGCAAGC TCTTCACGGA TGCCTCATCC 780CGAAGCATCG GGGCCCTCAA CAAGATCAAC TTCAACACCC GATTCGTCAT GAAGACGCTC 840ATGACCATCT GCCCGGGCAC GGTGCTGCTA ATGTTCAGCA TCTCTCTGTG GATCATCGCT 900GCCTGGACTG TGAGAGTCTG TGAAAGGTAC CATGACCAGC AGGACGTAAC TAGTAACTTT 960CTGGGTGCCA TGTGGCTCAT CTCCATCACG TTCCTTTCCA TTGGCTATGG GGACATGGTG 1020CCCCACACAT ACTGTGGGAA AGGTGTCTGT CTTCTCACTG GCATCATGGG TGCAGGCTGC 1080ACTGCCCTCG TGGTAGCTGT GGTTGCCCGG AAGCTCGAAC TCACCAAAGC AGAGAAGCAT 1140GTGCACAACT TCATGATGGA CACTCAGCTC ACCAAACGGA TCAAGAACGC TGCCGCCAAT 1200GTCCTCCGGG AAACATGGCT GATCTACAAA CACACAAAGC TGCTAAAGAA GATTGACCAC 1260GCCAAAGTCA GGAAACACCA GAGGAAGTTC CTCCAAGCTA TTCACCAACT GAGGGGTGTC 1320AAGATGGAAC AAAGGAAGCT GAGTGACCAA GCCAACACCC TGGTGGACCT TTCCAAGATG 1380CAGAACGTCA TGTATGACTT GATCACGGAG CTCAACGACC GGAGTGAAGA CCTGGAAAAG 1440CAGATTGGCA GCCTGGAATC CAAGCTGGAG CACCTCACAG CCAGCTTCAA TTCCCTGCCC 1500CTGCTCATCG CAGACACCCT GCGCCAACAG CAGCAGCAGC TGCTCACTGC CTTCGTGGAG 1560GCCCGGGGCA TCAGTGTGGC TGTGGGAACT AGCCACGCCC CTCCCTCTGA CAGCCCTATC 1620GGGATCAGCT CCACCTCTTT CCCGGAATTC CTAATATTC 1659 10 amino acids aminoacid <Unknown> linear peptide 17 Leu Ser Asp Tyr Ala Leu Ile Phe Gly Met1 5 10 10 amino acids amino acid <Unknown> linear peptide 18 Gln Arg LysPhe Leu Gln Ala Ile His Gln 1 5 10 579 amino acids amino acid <Unknown>linear protein Protein 1..579 /note= “human small conductance,calcium-activated potassium channel protein 2 (hSK2)” Region 134..461/note= “core region of hSK2” 19 Met Ser Ser Cys Arg Tyr Asn Gly Gly ValMet Arg Pro Leu Ser Asn 1 5 10 15 Leu Ser Ala Ser Arg Arg Asn Leu HisGlu Met Asp Ser Glu Ala Gln 20 25 30 Pro Leu Gln Pro Pro Ala Ser Val GlyGly Gly Gly Gly Ala Ser Ser 35 40 45 Pro Ser Ala Ala Ala Ala Ala Ala AlaAla Val Ser Ser Ser Ala Pro 50 55 60 Glu Ile Val Val Ser Lys Pro Glu HisAsn Asn Ser Asn Asn Leu Ala 65 70 75 80 Leu Tyr Gly Thr Gly Gly Gly GlySer Thr Gly Gly Gly Gly Gly Gly 85 90 95 Gly Gly Ser Gly His Gly Ser SerSer Gly Thr Lys Ser Ser Lys Lys 100 105 110 Lys Asn Gln Asn Ile Gly TyrLys Leu Gly His Arg Arg Ala Leu Phe 115 120 125 Glu Lys Arg Lys Arg LeuSer Asp Tyr Ala Leu Ile Phe Gly Met Phe 130 135 140 Gly Ile Val Val MetVal Ile Glu Thr Glu Leu Ser Trp Gly Ala Tyr 145 150 155 160 Asp Lys AlaSer Leu Tyr Ser Leu Ala Leu Lys Cys Leu Ile Ser Leu 165 170 175 Ser ThrIle Ile Leu Leu Gly Leu Ile Ile Val Tyr His Ala Arg Glu 180 185 190 IleGln Leu Phe Met Val Asp Asn Gly Ala Asp Asp Trp Arg Ile Ala 195 200 205Met Thr Tyr Glu Arg Ile Phe Phe Ile Cys Leu Glu Ile Leu Val Cys 210 215220 Ala Ile His Pro Ile Pro Gly Asn Tyr Thr Phe Thr Trp Thr Ala Arg 225230 235 240 Leu Ala Phe Ser Tyr Ala Pro Ser Thr Thr Thr Ala Asp Val AspIle 245 250 255 Ile Leu Ser Ile Pro Met Phe Leu Arg Leu Tyr Leu Ile AlaArg Val 260 265 270 Met Leu Leu His Ser Lys Leu Phe Thr Asp Ala Ser SerArg Ser Ile 275 280 285 Gly Ala Leu Asn Lys Ile Asn Phe Asn Thr Arg PheVal Met Lys Thr 290 295 300 Leu Met Thr Ile Cys Pro Gly Thr Val Leu LeuVal Phe Ser Ile Ser 305 310 315 320 Leu Trp Ile Ile Ala Ala Trp Thr ValArg Ala Cys Glu Arg Tyr His 325 330 335 Asp Gln Gln Asp Val Thr Ser AsnPhe Leu Gly Ala Met Trp Leu Ile 340 345 350 Ser Ile Thr Phe Leu Ser IleGly Tyr Gly Asp Met Val Pro Asn Thr 355 360 365 Tyr Cys Gly Lys Gly ValCys Leu Leu Thr Gly Ile Met Gly Ala Gly 370 375 380 Cys Thr Ala Leu ValVal Ala Val Val Ala Arg Lys Leu Glu Leu Thr 385 390 395 400 Lys Ala GluLys His Val His Asn Phe Met Met Asp Thr Gln Leu Thr 405 410 415 Lys ArgVal Lys Asn Ala Ala Ala Asn Val Leu Arg Glu Thr Trp Leu 420 425 430 IleTyr Lys Asn Thr Lys Leu Val Lys Lys Ile Asp His Ala Lys Val 435 440 445Arg Lys His Gln Arg Lys Phe Leu Gln Ala Ile His Gln Leu Arg Ser 450 455460 Val Lys Met Glu Gln Arg Lys Leu Asn Asp Gln Ala Asn Thr Leu Val 465470 475 480 Asp Leu Ala Lys Thr Gln Asn Ile Met Tyr Asp Met Ile Ser AspLeu 485 490 495 Asn Glu Arg Ser Glu Asp Phe Glu Lys Arg Ile Val Thr LeuGlu Thr 500 505 510 Lys Leu Glu Thr Leu Ile Gly Ser Ile His Ala Leu ProGly Leu Ile 515 520 525 Ser Gln Thr Ile Arg Gln Gln Gln Arg Asp Phe IleGlu Ala Gln Met 530 535 540 Glu Ser Tyr Asp Lys His Val Thr Tyr Asn AlaGlu Arg Ser Arg Ser 545 550 555 560 Ser Ser Arg Arg Arg Arg Ser Ser SerThr Ala Pro Pro Thr Ser Ser 565 570 575 Glu Ser Ser 557 amino acidsamino acid <Unknown> linear protein Protein 1..557 /note= “N-terminallytruncated form of human small conductance, calcium-activated potassiumchannel protein 3 (hSK3)” Region 109..436 /note= “core region of hSK3”20 Met Ser Ser Cys Lys Tyr Ser Gly Gly Val Met Lys Pro Leu Ser Arg 1 510 15 Leu Ser Ala Ser Arg Arg Asn Leu Ile Glu Ala Glu Thr Glu Gly Gln 2025 30 Pro Leu Gln Leu Phe Ser Pro Ser Asn Pro Pro Glu Ile Val Ile Ser 3540 45 Ser Arg Glu Asp Asn His Ala His Gln Thr Leu Leu His His Pro Asn 5055 60 Ala Thr His Asn His Gln His Ala Gly Thr Thr Ala Ser Ser Thr Thr 6570 75 80 Phe Pro Lys Ala Asn Lys Arg Lys Asn Gln Asn Ile Gly Tyr Lys Leu85 90 95 Gly His Arg Arg Ala Leu Phe Glu Lys Arg Lys Arg Leu Ser Asp Tyr100 105 110 Ala Leu Ile Phe Gly Met Phe Gly Ile Val Val Met Val Ile GluThr 115 120 125 Glu Leu Ser Trp Gly Leu Tyr Ser Lys Asp Ser Met Phe SerLeu Ala 130 135 140 Leu Lys Cys Leu Ile Ser Leu Ser Thr Ile Ile Leu LeuGly Leu Ile 145 150 155 160 Ile Ala Tyr His Thr Arg Glu Val Gln Leu PheVal Ile Asp Asn Gly 165 170 175 Ala Asp Asp Trp Arg Ile Ala Met Thr TyrGlu Arg Ile Leu Tyr Ile 180 185 190 Ser Leu Glu Met Leu Val Cys Ala IleHis Pro Ile Pro Gly Glu Tyr 195 200 205 Lys Phe Phe Trp Thr Ala Arg LeuAla Phe Ser Tyr Thr Pro Ser Arg 210 215 220 Ala Glu Ala Asp Val Asp IleIle Leu Ser Ile Pro Met Phe Leu Arg 225 230 235 240 Leu Tyr Leu Ile AlaArg Val Met Leu Leu His Ser Lys Leu Phe Thr 245 250 255 Asp Ala Ser SerArg Ser Ile Gly Ala Leu Asn Lys Ile Asn Phe Asn 260 265 270 Thr Arg PheVal Met Lys Thr Leu Met Thr Ile Cys Pro Gly Thr Val 275 280 285 Leu LeuVal Phe Ser Ile Ser Leu Trp Ile Ile Ala Ala Trp Thr Val 290 295 300 ArgVal Cys Glu Arg Tyr His Asp Gln Gln Asp Val Thr Ser Asn Phe 305 310 315320 Leu Gly Ala Met Trp Leu Ile Ser Ile Thr Phe Leu Ser Ile Gly Tyr 325330 335 Gly Asp Met Val Pro His Thr Tyr Cys Gly Lys Gly Val Cys Leu Leu340 345 350 Thr Gly Ile Met Gly Ala Gly Cys Thr Ala Leu Val Val Ala ValVal 355 360 365 Ala Arg Lys Leu Glu Leu Thr Lys Ala Glu Lys His Val HisAsn Phe 370 375 380 Met Met Asp Thr Gln Leu Thr Lys Arg Ile Lys Asn AlaAla Ala Asn 385 390 395 400 Val Leu Arg Glu Thr Trp Leu Ile Tyr Lys HisThr Lys Leu Leu Lys 405 410 415 Lys Ile Asp His Ala Lys Val Arg Lys HisGln Arg Lys Phe Leu Gln 420 425 430 Ala Ile His Gln Leu Arg Ser Val LysMet Glu Gln Arg Lys Leu Ser 435 440 445 Asp Gln Ala Asn Thr Leu Val AspLeu Ser Lys Met Gln Asn Val Met 450 455 460 Tyr Asp Leu Ile Thr Glu LeuAsn Asp Arg Ser Glu Asp Leu Glu Lys 465 470 475 480 Gln Ile Gly Ser LeuGlu Ser Lys Leu Glu His Leu Thr Ala Ser Phe 485 490 495 Asn Ser Leu ProLeu Leu Ile Ala Asp Thr Leu Arg Gln Gln Gln Gln 500 505 510 Gln Leu LeuSer Ala Ile Ile Glu Ala Arg Gly Val Ser Val Ala Val 515 520 525 Gly ThrThr His Thr Pro Ile Ser Asp Ser Pro Ile Gly Val Ser Ser 530 535 540 ThrSer Phe Pro Thr Pro Tyr Thr Ser Ser Ser Ser Cys 545 550 555 1740 basepairs nucleic acid single linear cDNA - 1..1740 /note= “human smallconductance, calcium-activated potassium channel protein 2 (hSK2) cDNA”21 ATGAGCAGCT GCAGGTACAA CGGGGGCGTC ATGCGGCCGC TCAGCAACTT GAGCGCGTCC 60CGCCGGAACC TGCACGAGAT GGACTCAGAG GCGCAGCCCC TGCAGCCCCC CGCGTCTGTC 120GGAGGAGGTG GCGGCGCGTC CTCCCCGTCT GCAGCCGCTG CCGCCGCCGC CGCTGTTTCG 180TCCTCAGCCC CCGAGATCGT GGTGTCTAAG CCCGAGCACA ACAACTCCAA CAACCTGGCG 240CTCTATGGAA CCGGCGGCGG AGGCAGCACT GGAGGAGGCG GCGGCGGTGG CGGGAGCGGG 300CACGGCAGCA GCAGTGGCAC CAAGTCCAGC AAAAAGAAAA ACCAGAACAT CGGCTACAAG 360CTGGGCCACC GGCGCGCCCT GTTCGAAAAG CGCAAGCGGC TCAGCGACTA CGCGCTCATC 420TTCGGCATGT TCGGCATCGT GGTCATGGTC ATCGAGACCG AGCTGTCGTG GGGCGCCTAC 480GACAAGGCGT CGCTGTATTC CTTAGCTCTG AAATGCCTTA TCAGTCTCTC CACGATCATC 540CTGCTCGGTC TGATCATCGT GTACCACGCC AGGGAAATAC AGTTGTTCAT GGTGGACAAT 600GGAGCAGATG ACTGGAGAAT AGCCATGACT TATGAGCGTA TTTTCTTCAT CTGCTTGGAA 660ATACTGGTGT GTGCTATTCA TCCCATACCT GGGAATTATA CATTCACATG GACGGCCCGG 720CTTGCCTTCT CCTATGCCCC ATCCACAACC ACCGCTGATG TGGATATTAT TTTATCTATA 780CCAATGTTCT TAAGACTCTA TCTGATTGCC AGAGTCATGC TTTTACATAG CAAACTTTTC 840ACTGATGCCT CCTCTAGAAG CATTGGAGCA CTTAATAAGA TAAACTTCAA TACACGTTTT 900GTTATGAAGA CTTTAATGAC TATATGCCCA GGAACTGTAC TCTTGGTTTT TAGTATCTCA 960TTATGGATAA TTGCCGCATG GACTGTCCGA GCTTGTGAAA GGTACCATGA TCAACAGGAT 1020GTTACTAGCA ACTTCCTTGG AGCGATGTGG TTGATATCAA TAACTTTTCT CTCCATTGGT 1080TATGGTGACA TGGTACCTAA CACATACTGT GGAAAAGGAG TCTGCTTACT TACTGGAATT 1140ATGGGTGCTG GTTGCACAGC CCTGGTGGTA GCTGTAGTGG CAAGGAAGCT AGAACTTACC 1200AAAGCAGAAA AACACGTGCA CAATTTCATG ATGGATACTC AGCTGACTAA AAGAGTAAAA 1260AATGCAGCTG CCAATGTACT CAGGGAAACA TGGCTAATTT ACAAAAATAC AAAGCTAGTG 1320AAAAAGATAG ATCATGCAAA AGTAAGAAAA CATCAACGAA AATTCCTGCA AGCTATTCAT 1380CAATTAAGAA GTGTAAAAAT GGAACAGAGG AAACTGAATG ACCAAGCAAA CACTTTGGTG 1440GACTTGGCAA AGACCCAGAA CATCATGTAT GATATGATTT CTGACTTAAA CGAAAGGAGT 1500GAAGACTTCG AGAAGAGGAT TGTTACCCTG GAAACAAAAT TAGAGACTTT GATTGGTAGC 1560ATCCACGCCC TCCCTGGGCT CATAAGCCAG ACCATCAGGC AGCAGCAGAG AGATTTCATT 1620GAGGCTCAGA TGGAGAGCTA CGACAAGCAC GTCACTTACA ATGCTGAGCG GTCCCGGTCC 1680TCGTCCAGGA GGCGGCGGTC CTCTTCCACA GCACCACCAA CTTCATCAGA GAGTAGCTAG 17401674 base pairs nucleic acid single linear cDNA - 1..1674 /note=“N-terminally truncated cDNA for human small conductance,calcium-activated potassium channel protein 3 (hSK3)” 22 ATGAGCTCCTGCAAGTATAG CGGTGGGGTC ATGAAGCCCC TCAGCCGCCT CAGCGCCTCC 60 CGGAGGAACCTCATCGAGGC CGAGACTGAG GGCCAACCCC TCCAGCTTTT CAGCCCTAGC 120 AACCCCCCGGAGATCGTCAT CTCCTCCCGG GAGGACAACC ATGCCCACCA GACCCTGCTC 180 CATCACCCTAATGCCACCCA CAACCACCAG CATGCCGGCA CCACCGCCAG CAGCACCACC 240 TTCCCCAAAGCCAACAAGCG GAAAAACCAA AACATTGGCT ATAAGCTGGG ACACAGGAGG 300 GCCCTGTTTGAAAAGAGAAA GCGACTGAGT GACTATGCTC TGATTTTTGG GATGTTTGGA 360 ATTGTTGTTATGGTGATAGA GACCGAGCTC TCTTGGGGTT TGTACTCAAA GGACTCCATG 420 TTTTCGTTGGCCCTGAAATG CCTTATCAGT CTGTCCACCA TCATCCTTTT GGGCTTGATC 480 ATCGCCTACCACACACGTGA AGTCCAGCTC TTCGTGATCG ACAACGGCGC GGATGACTGG 540 CGGATAGCCATGACCTACGA GCGCATCCTC TACATCAGCC TGGAGATGCT GGTGTGCGCC 600 ATCCACCCCATTCCTGGCGA GTACAAGTTC TTCTGGACGG CACGCCTGGC CTTCTCCTAC 660 ACACCCTCCCGGGCGGAGGC CGATGTGGAC ATCATCCTGT CTATCCCCAT GTTCCTGCGC 720 CTGTACCTGATCGCCCGAGT CATGCTGCTG CACAGCAAGC TCTTCACCGA TGCCTCGTCC 780 CGCAGCATCGGGGCCCTCAA CAAGATCAAC TTCAACACCC GCTTTGTCAT GAAGACGCTC 840 ATGACCATCTGCCCTGGCAC TGTGCTGCTC GTGTTCAGCA TCTCTCTGTG GATCATTGCT 900 GCCTGGACCGTCCGTGTCTG TGAAAGGTAC CATGACCAGC AGGACGTAAC TAGTAACTTT 960 CTGGGTGCCATGTGGCTCAT CTCCATCACA TTCCTTTCCA TTGGTTATGG GGACATGGTG 1020 CCCCACACATACTGTGGGAA AGGTGTCTGT CTCCTCACTG GCATCATGGG TGCAGGCTGC 1080 ACTGCCCTTGTGGTGGCCGT GGTGGCCCGA AAGCTGGAAC TCACCAAAGC GGAGAAGCAC 1140 GTTCATAACTTCATGATGGA CACTCAGCTC ACCAAGCGGA TCAAGAATGC TGCAGCCAAT 1200 GTCCTTCGGGAAACATGGTT AATCTATAAA CACACAAAGC TGCTAAAGAA GATTGACCAT 1260 GCCAAAGTGAGGAAACACCA GAGGAAGTTC CTCCAAGCTA TCCACCAGTT GAGGAGCGTC 1320 AAGATGGAACAGAGGAAGCT GAGTGACCAA GCCAACACTC TGGTGGACCT TTCCAAGATG 1380 CAGAATGTCATGTATGACTT AATCACAGAA CTCAATGACC GGAGCGAAGA CCTGGAGAAG 1440 CAGATTGGCAGCCTGGAGTC GAAGCTGGAG CATCTCACCG CCAGCTTCAA CTCCCTGCCG 1500 CTGCTCATCGCCGACACCCT GCGCCAGCAG CAGCAGCAGC TCCTGTCTGC CATCATCGAG 1560 GCCCGGGGTGTCAGCGTGGC AGTGGGCACC ACCCACACCC CAATCTCCGA TAGCCCCATT 1620 GGGGTCAGCTCCACCTCCTT CCCGACCCCG TACACAAGTT CAAGCAGTTG CTAA 1674 22 base pairsnucleic acid single linear DNA 23 ATGAGCAGCT GCAGGTACAA CG 22 23 basepairs nucleic acid single linear DNA 24 CTAGCTACTC TCTGATGAAG TTG 23 21base pairs nucleic acid single linear DNA 25 ATGAGCTCCT GCAAGTATAG C 2122 base pairs nucleic acid single linear DNA 26 TTAGCAACTG CTTGAACTTG TG22 328 amino acids amino acid <Unknown> linear peptide Region 1..328/note= “core region of hSK1 from amino acid positions 124 through 451”27 Leu Ser Asp Tyr Ala Leu Ile Phe Gly Met Phe Gly Ile Val Val Met 1 510 15 Val Thr Glu Thr Glu Leu Ser Trp Gly Val Tyr Thr Lys Glu Ser Leu 2025 30 Tyr Ser Phe Ala Leu Lys Cys Leu Ile Ser Leu Ser Thr Ala Ile Leu 3540 45 Leu Gly Leu Val Val Leu Tyr His Ala Arg Glu Ile Gln Leu Phe Met 5055 60 Val Asp Asn Gly Ala Asp Asp Trp Arg Ile Ala Met Thr Cys Glu Arg 6570 75 80 Val Phe Leu Ile Ser Leu Glu Leu Ala Val Cys Ala Ile His Pro Val85 90 95 Pro Gly His Tyr Arg Phe Thr Trp Thr Ala Arg Leu Ala Phe Thr Tyr100 105 110 Ala Pro Ser Val Ala Glu Ala Asp Val Asp Val Leu Leu Ser IlePro 115 120 125 Met Phe Leu Arg Leu Tyr Leu Leu Gly Arg Val Met Leu LeuHis Ser 130 135 140 Lys Ile Phe Thr Asp Ala Ser Ser Arg Ser Ile Gly AlaLeu Asn Lys 145 150 155 160 Ile Thr Phe Asn Thr Arg Phe Val Met Lys ThrLeu Met Thr Ile Cys 165 170 175 Pro Gly Thr Val Leu Leu Val Phe Ser IleSer Ser Trp Ile Ile Ala 180 185 190 Ala Trp Thr Val Arg Val Cys Glu ArgTyr His Asp Lys Gln Glu Val 195 200 205 Thr Ser Asn Phe Leu Gly Ala MetTrp Leu Ile Ser Ile Thr Phe Leu 210 215 220 Ser Ile Gly Tyr Gly Asp MetVal Pro His Thr Tyr Cys Gly Lys Gly 225 230 235 240 Val Cys Leu Leu ThrGly Ile Met Gly Ala Gly Cys Thr Ala Leu Val 245 250 255 Val Ala Val ValAla Arg Lys Leu Glu Leu Thr Lys Ala Glu Lys His 260 265 270 Val His AsnPhe Met Met Asp Thr Gln Leu Thr Lys Arg Val Lys Asn 275 280 285 Ala AlaAla Asn Val Leu Arg Glu Thr Trp Leu Ile Tyr Lys His Thr 290 295 300 ArgLeu Val Lys Lys Pro Asp Gln Ala Arg Val Arg Lys His Gln Arg 305 310 315320 Lys Phe Leu Gln Ala Ile His Gln 325 16 amino acids amino acid<Unknown> linear peptide 28 Gly His Arg Arg Ala Leu Phe Glu Lys Arg LysArg Leu Ser Asp Tyr 1 5 10 15 12 amino acids amino acid <Unknown> linearpeptide 29 Phe Thr Asp Ala Ser Ser Arg Ser Ile Gly Ala Leu 1 5 10 25amino acids amino acid <Unknown> linear peptide 30 Ala Arg Lys Leu GluLeu Thr Lys Ala Glu Lys His Val His Asn Phe 1 5 10 15 Met Met Asp ThrGln Leu Thr Lys Arg 20 25 1287 base pairs nucleic acid single linearcDNA - 1..1287 /note= “human intermediate conductance, calcium-activatedpotassium channel protein 1 (hIK1) cDNA” 31 ATGGGCGGGG ATCTGGTGCTTGGCCTGGGG GCCTTGAGAC GCCGAAAGCG CTTGCTGGAG 60 CAGGAGAAGT CTCTGGCCGGCTGGGCACTG GTGCTGGCAG GAACTGGCAT TGGACTCATG 120 GTGCTGCATG CAGAGATGCTGTGGTTCGGG GGGTGCTCGT GGGCGCTCTA CCTGTTCCTG 180 GTTAAATGCA CGATCAGCATTTCCACCTTC TTACTCCTCT GCCTCATCGT GGCCTTTCAT 240 GCCAAAGAGG TCCAGCTGTTCAGTACCGAC AACGGGCTGC GGGACTGGCG CGTGGTGCTC 300 CTGACCGGGC GGCAGGCGGCGCAGATCGTG CTGGAGCTGG TGGTGTGTGG GCTGCACCCG 360 GCGCCCGTGC GGGGCCCGCCGTGCGTGCAG GATTTAGGGG CGCCGCTGAC CTCCCCGCAG 420 CCCTGGCCGG GATTCCTGGGCCAAGGGGAA GCGCTGCTGT CCCTGGCCAT GCTGCTGCGT 480 CTCTACCTGG TGCCCCGCGCCGTGCTCCTG CGCAGCGGCG TCCTGCTCAA CGCTTCCTAC 540 CGCAGCATCG GCGCTCTCAATCAAGTCCGC TTCCGCCACT GGTTCGTGGC CAAGCTTTAC 600 ATGAACACGC ACCCTGGCCGCCTGCTGCTC GGCCTCACGC TTGGCCTCTG GCTGACCACC 660 GCCTGGGTGC TGTCCGTGGCCGAGAGGCAG GCTGTTAATG CCACTGGGCA CCTTTCAGAC 720 ACACTTTGGC TGATCCCCATCACATTCCTG ACCATCGGCT ATGGTGACGT GGTGCCGGGC 780 ACCATGTTGG GCAAGATCGTCTGCCTGTGC ACTGGAGTCA TGGGTGTCTG CTGCACAGCC 840 CTGCTGGTGG CCGTGGTGGCCCGGAAGCTG GAGTTTAACA AGGCAGAGAA GCACGTGCAC 900 AACTTCATGA TGGATATCCAGAATACCAAA GAGATGAAGG AGTCCGCTGC CCGAGTGCTA 960 CAAGAAGCCT GGATGTTCTACAAACATACT CGCAGGAAGG AGTCTCATGC TGCCCGCAGG 1020 CATCAGCGCA AGCTGCTGGCCGCCATCAAC GCGTTCCGCC AGGTGCGGCT GAAACACCGG 1080 AAGCTCCGGG AACAAGTGAACTCCATGGTG GACATCTCCA AGATGCACAT GATCCTGTAT 1140 GACCTGCAGC AGAATCTGAGCAGCTCACAC CGGGCCCTGG AGAAACAGAT TGACACGCTG 1200 GCGGGGAAGC TGGATGCCCTGACTGAGCTG CTTAGCACTG CCCTGGGGCC GAGGCAGCTT 1260 CCAGAACCCA GCCAGCAGTCCAAGTAG 1287 428 amino acids amino acid <Unknown> linear protein Protein1..428 /note= “human intermediate conductance, calcium-activatedpotassium channel protein 1 (hIK1)” Region 25..351 /note= “core regionof hIK1” 32 Met Gly Gly Asp Leu Val Leu Gly Leu Gly Ala Leu Arg Arg ArgLys 1 5 10 15 Arg Leu Leu Glu Gln Glu Lys Ser Leu Ala Gly Trp Ala LeuVal Leu 20 25 30 Ala Gly Thr Gly Ile Gly Leu Met Val Leu His Ala Glu MetLeu Trp 35 40 45 Phe Gly Gly Cys Ser Trp Ala Leu Tyr Leu Phe Leu Val LysCys Thr 50 55 60 Ile Ser Ile Ser Thr Phe Leu Leu Leu Cys Leu Ile Val AlaPhe His 65 70 75 80 Ala Lys Glu Val Gln Leu Phe Ser Thr Asp Asn Gly LeuArg Asp Trp 85 90 95 Arg Val Val Leu Leu Thr Gly Arg Gln Ala Ala Gln IleVal Leu Glu 100 105 110 Leu Val Val Cys Gly Leu His Pro Ala Pro Val ArgGly Pro Pro Cys 115 120 125 Val Gln Asp Leu Gly Ala Pro Leu Thr Ser ProGln Pro Trp Pro Gly 130 135 140 Phe Leu Gly Gln Gly Glu Ala Leu Leu SerLeu Ala Met Leu Leu Arg 145 150 155 160 Leu Tyr Leu Val Pro Arg Ala ValLeu Leu Arg Ser Gly Val Leu Leu 165 170 175 Asn Ala Ser Tyr Arg Ser IleGly Ala Leu Asn Gln Val Arg Phe Arg 180 185 190 His Trp Phe Val Ala LysLeu Tyr Met Asn Thr His Pro Gly Arg Leu 195 200 205 Leu Leu Gly Leu ThrLeu Gly Leu Trp Leu Thr Thr Ala Trp Val Leu 210 215 220 Ser Val Ala GluArg Gln Ala Val Asn Ala Thr Gly His Leu Ser Asp 225 230 235 240 Thr LeuTrp Leu Ile Pro Ile Thr Phe Leu Thr Ile Gly Tyr Gly Asp 245 250 255 ValVal Pro Gly Thr Met Leu Gly Lys Ile Val Cys Leu Cys Thr Gly 260 265 270Val Met Gly Val Cys Cys Thr Ala Leu Leu Val Ala Val Val Ala Arg 275 280285 Lys Leu Glu Phe Asn Lys Ala Glu Lys His Val His Asn Phe Met Met 290295 300 Asp Ile Gln Asn Thr Lys Glu Met Lys Glu Ser Ala Ala Arg Val Leu305 310 315 320 Gln Glu Ala Trp Met Phe Tyr Lys His Thr Arg Arg Lys GluSer His 325 330 335 Ala Ala Arg Arg His Gln Arg Lys Leu Leu Ala Ala IleAsn Ala Phe 340 345 350 Arg Gln Val Arg Leu Lys His Arg Lys Leu Arg GluGln Val Asn Ser 355 360 365 Met Val Asp Ile Ser Lys Met His Met Ile LeuTyr Asp Leu Gln Gln 370 375 380 Asn Leu Ser Ser Ser His Arg Ala Leu GluLys Gln Ile Asp Thr Leu 385 390 395 400 Ala Gly Lys Leu Asp Ala Leu ThrGlu Leu Leu Ser Thr Ala Leu Gly 405 410 415 Pro Arg Gln Leu Pro Glu ProSer Gln Gln Ser Lys 420 425 30 amino acids amino acid <Unknown> linearpeptide 33 Val Arg Gly Pro Pro Cys Val Gln Asp Leu Gly Ala Pro Leu ThrSer 1 5 10 15 Pro Gln Pro Trp Pro Gly Phe Leu Gly Gln Gly Glu Ala Leu 2025 30 21 base pairs nucleic acid single linear DNA 34 GCCGTGCGTGCAGGATTTAG G 21 21 base pairs nucleic acid single linear DNA 35CCAGAGGCCA AGCGTGAGGC C 21 21 base pairs nucleic acid single linear DNA36 TCCAAGATGC ACATGATCCT G 21 21 base pairs nucleic acid single linearDNA 37 GGACTGCTGG CTGGGTTCTG G 21 22 base pairs nucleic acid singlelinear DNA 38 ATGGGCGGGG ATCTGGTGCT TG 22 24 base pairs nucleic acidsingle linear DNA 39 CTACTTGGAC TGCTGGCTGG GTTC 24 23 base pairs nucleicacid single linear DNA 40 ATGGGCGGGG ATCTGGTGCT TGG 23 23 base pairsnucleic acid single linear DNA 41 GGGTCCAGCT ACTTGGACTG CTG 23 24 aminoacids amino acid <Unknown> linear peptide 42 Ala Arg Lys Leu Glu Leu ThrLys Ala Glu Lys His Val His Asn Phe 1 5 10 15 Met Met Asp Thr Gln LeuThr Lys 20 732 amino acids amino acid <Unknown> linear protein Protein1..732 /note= “full-length rat small conductance, calcium-activatedpotassium channel protein 3 (rSK3)” 43 Met Asp Thr Ser Gly His Phe HisGlu Ser Gly Val Gly Asp Leu Asp 1 5 10 15 Glu Asp Pro Lys Cys Pro CysPro Ser Ser Gly Asp Glu Gln Gln Gln 20 25 30 Gln Gln Gln Pro Pro Pro ProSer Ala Pro Pro Ala Val Pro Gln Gln 35 40 45 Pro Pro Gly Pro Leu Leu GlnPro Gln Pro Pro Gln Leu Gln Gln Gln 50 55 60 Gln Gln Gln Gln Gln Gln GlnGln Gln Gln Gln Gln Gln Gln Gln Gln 65 70 75 80 Ala Pro Leu His Pro LeuPro Gln Leu Ala Gln Leu Gln Ser Gln Val 85 90 95 Val His Pro Gly Leu LeuHis Ser Ser Pro Thr Ala Phe Arg Ala Pro 100 105 110 Asn Ser Ala Asn SerThr Ala Ile Leu His Pro Ser Ser Arg Gln Gly 115 120 125 Ser Gln Leu AsnLeu Asn Asp His Leu Val Gly His Ser Pro Ser Ser 130 135 140 Thr Ala ThrSer Gly Pro Gly Gly Gly Ser Arg His Arg Gln Ala Ser 145 150 155 160 ProVal Val His Arg Arg Asp Ser Asn Pro Phe Thr Glu Ile Ala Met 165 170 175Ser Ser Cys Lys Tyr Ser Gly Gly Val Met Lys Pro Leu Ser Arg Leu 180 185190 Ser Ala Ser Arg Arg Asn Leu Ile Glu Ala Glu Pro Glu Gly Gln Pro 195200 205 Leu Gln Leu Phe Ser Pro Ser Asn Pro Pro Glu Ile Ile Ile Ser Ser210 215 220 Arg Glu Asp Asn His Ala His Gln Thr Leu Leu His His Pro AsnAla 225 230 235 240 Thr His Asn His Gln His Ala Gly Thr Thr Ala Gly SerThr Thr Phe 245 250 255 Pro Lys Ala Asn Lys Arg Lys Asn Gln Asn Ile GlyTyr Lys Leu Gly 260 265 270 His Arg Arg Ala Leu Phe Glu Lys Arg Lys ArgLeu Ser Asp Tyr Ala 275 280 285 Leu Ile Phe Gly Met Phe Gly Ile Val ValMet Val Ile Glu Thr Glu 290 295 300 Leu Ser Trp Gly Leu Tyr Ser Lys AspSer Met Phe Ser Leu Ala Leu 305 310 315 320 Lys Cys Leu Ile Ser Leu SerThr Ile Ile Leu Leu Gly Leu Ile Ile 325 330 335 Ala Tyr His Thr Arg GluVal Gln Leu Phe Val Ile Asp Asn Gly Ala 340 345 350 Asp Asp Trp Arg IleAla Met Thr Tyr Glu Arg Ile Leu Tyr Ile Ser 355 360 365 Leu Glu Met LeuVal Cys Ala Ile His Pro Ile Pro Gly Glu Tyr Lys 370 375 380 Phe Phe TrpThr Ala Arg Leu Ala Phe Ser Tyr Thr Pro Ser Arg Ala 385 390 395 400 GluAla Asp Val Asp Ile Ile Leu Ser Ile Pro Met Phe Leu Arg Leu 405 410 415Tyr Leu Ile Ala Arg Val Met Leu Leu His Ser Lys Leu Phe Thr Asp 420 425430 Ala Ser Ser Arg Ser Ile Gly Ala Leu Asn Lys Ile Asn Phe Asn Thr 435440 445 Arg Phe Val Met Lys Thr Leu Met Thr Ile Cys Pro Gly Thr Val Leu450 455 460 Leu Met Phe Ser Ile Ser Leu Trp Ile Ile Ala Ala Trp Thr ValArg 465 470 475 480 Val Cys Glu Arg Tyr His Asp Gln Gln Asp Val Thr SerAsn Phe Leu 485 490 495 Gly Ala Met Trp Leu Ile Ser Ile Thr Phe Leu SerIle Gly Tyr Gly 500 505 510 Asp Met Val Pro His Thr Tyr Cys Gly Lys GlyVal Cys Leu Leu Thr 515 520 525 Gly Ile Met Gly Ala Gly Cys Thr Ala LeuVal Val Ala Val Val Ala 530 535 540 Arg Lys Leu Glu Leu Thr Lys Ala GluLys His Val His Asn Phe Met 545 550 555 560 Met Asp Thr Gln Leu Thr LysArg Ile Lys Asn Ala Ala Ala Asn Val 565 570 575 Leu Arg Glu Thr Trp LeuIle Tyr Lys His Thr Lys Leu Leu Lys Lys 580 585 590 Ile Asp His Ala LysVal Arg Lys His Gln Arg Lys Phe Leu Gln Ala 595 600 605 Ile His Gln LeuArg Gly Val Lys Met Glu Gln Arg Lys Leu Ser Asp 610 615 620 Gln Ala AsnThr Leu Val Asp Leu Ser Lys Met Gln Asn Val Met Tyr 625 630 635 640 AspLeu Ile Thr Glu Leu Asn Asp Arg Ser Glu Asp Leu Glu Lys Gln 645 650 655Ile Gly Ser Leu Glu Ser Lys Leu Glu His Leu Thr Ala Ser Phe Asn 660 665670 Ser Leu Pro Leu Leu Ile Ala Asp Thr Leu Arg Gln Gln Gln Gln Gln 675680 685 Leu Leu Thr Ala Phe Val Glu Ala Arg Gly Ile Ser Val Ala Val Gly690 695 700 Thr Ser His Ala Pro Pro Ser Asp Ser Pro Ile Gly Ile Ser SerThr 705 710 715 720 Ser Phe Pro Thr Pro Tyr Thr Ser Ser Ser Ser Cys 725730 2224 base pairs nucleic acid single linear cDNA - 1..2224 /note=“rat small conductance, calcium-activated potassium channel protein 3(rSK3) full-length cDNA” 44 CATGGACACT TCTGGGCACT TCCATGAGTC GGGGGTGGGGGATCTGGATG AAGACCCCAA 60 GTGTCCCTGT CCATCTTCTG GGGACGAGCA ACAGCAGCAACAGCAACCGC CACCACCGTC 120 AGCGCCACCA GCAGTCCCCC AGCAGCCTCC GGGACCCTTGCTGCAGCCTC AGCCTCCGCA 180 GCTTCAGCAG CAGCAGCAGC AGCAGCAGCA GCAGCAGCAGCAGCAGCAGC AGCAGCAGCA 240 GGCTCCACTG CACCCCCTGC CTCAGCTTGC CCAACTCCAGAGCCAGGTTG TCCATCCTGG 300 TCTGTTGCAC TCTTCTCCCA CGGCTTTCAG GGCTCCCAATTCAGCCAACT CCACCGCCAT 360 CCTCCACCCT TCCTCCAGGC AAGGCAGCCA GCTAAATCTCAATGACCACT TGGTTGGCCA 420 CTCTCCAAGT TCCACAGCCA CAAGTGGGCC TGGTGGAGGCAGCCGGCACC GGCAGGCCAG 480 CCCCGTGGTG CACCGGCGGG ACAGCAATCC CTTCACGGAGATAGCTATGA GCTCCTGCAA 540 ATACAGCGGT GGGGTCATGA AGCCCCTCAG CCGCCTCAGCGCCTCTCGGA GAAACCTTAT 600 CGAGGCCGAG CCTGAGGGCC AACCCCTCCA GCTCTTCAGTCCCAGCAACC CCCCAGAGAT 660 TATCATCTCC TCCAGGGAGG ATAACCATGC CCACCAGACTCTGCTCCATC ACCCCAACGC 720 TACCCACAAC CACCAGCATG CCGGCACCAC TGCTGGCAGCACCACCTTCC CCAAAGCCAA 780 CAAGCGGAAA AACCAAAACA TTGGCTATAA GCTGGGGCACAGGAGGGCCC TGTTTGAAAA 840 GAGAAAGCGA CTGAGTGACT ATGCTCTGAT TTTTGGGATGTTTGGAATTG TTGTTATGGT 900 GATAGAGACC GAACTGTCTT GGGGTTTGTA CTCAAAGGATTCCATGTTTT CGTTGGCCCT 960 GAAATGCCTT ATCAGTTTAT CCACCATCAT CCTGCTTGGTTTGATCATCG CCTACCACAC 1020 AAGGGAAGTA CAGCTCTTTG TGATCGACAA TGGTGCAGATGACTGGCGGA TAGCCATGAC 1080 CTATGAGCGC ATCCTCTACA TCAGCCTGGA GATGCTGGTGTGCGCCATCC ACCCCATTCC 1140 TGGAGAGTAC AAGTTCTTCT GGACGGCACG CCTGGCCTTCTCCTACACCC CCTCTCGGGC 1200 AGAGGCTGAC GTGGACATTA TTCTGTCCAT CCCCATGTTCTTGCGCCTAT ACCTGATCGC 1260 CCGAGTCATG CTGCTACATA GCAAGCTCTT CACGGATGCCTCATCCCGAA GCATCGGGGC 1320 CCTCAACAAG ATCAACTTCA ACACCCGATT CGTCATGAAGACGCTCATGA CCATCTGCCC 1380 GGGCACGGTG CTGCTAATGT TCAGCATCTC TCTGTGGATCATCGCTGCCT GGACTGTGAG 1440 AGTCTGTGAA AGGTACCATG ACCAGCAGGA CGTAACTAGTAACTTTCTGG GTGCCATGTG 1500 GCTCATCTCC ATCACGTTCC TTTCCATTGG CTATGGGGACATGGTGCCCC ACACATACTG 1560 TGGGAAAGGT GTCTGTCTTC TCACTGGCAT CATGGGTGCAGGCTGCACTG CCCTCGTGGT 1620 AGCTGTGGTT GCCCGGAAGC TCGAACTCAC CAAAGCAGAGAAGCATGTGC ACAACTTCAT 1680 GATGGACACT CAGCTCACCA AACGGATCAA GAACGCTGCCGCCAATGTCC TCCGGGAAAC 1740 ATGGCTGATC TACAAACACA CAAAGCTGCT AAAGAAGATTGACCACGCCA AAGTCAGGAA 1800 ACACCAGAGG AAGTTCCTCC AAGCTATTCA CCAACTGAGGGGTGTCAAGA TGGAACAAAG 1860 GAAGCTGAGT GACCAAGCCA ACACCCTGGT GGACCTTTCCAAGATGCAGA ACGTCATGTA 1920 TGACTTGATC ACGGAGCTCA ACGACCGGAG TGAAGACCTGGAAAAGCAGA TTGGCAGCCT 1980 GGAATCCAAG CTGGAGCACC TCACAGCCAG CTTCAATTCCCTGCCCCTGC TCATCGCAGA 2040 CACCCTGCGC CAACAGCAGC AGCAGCTGCT CACTGCCTTCGTGGAGGCCC GGGGCATCAG 2100 TGTGGCTGTG GGAACTAGCC ACGCCCCTCC CTCTGACAGCCCTATCGGGA TCAGCTCCAC 2160 CTCTTTCCCA ACCCCATACA CAAGTTCAAG CAGTTGCTAAATAAAACTCC CCACTCCAGA 2220 AGCA 2224 25 amino acids amino acid <Unknown>linear peptide 45 Phe Xaa Ser Ile Pro Xaa Xaa Xaa Trp Trp Ala Xaa ValThr Met Thr 1 5 10 15 Thr Val Gly Tyr Gly Asp Met Xaa Pro 20 25 4 aminoacids amino acid <Unknown> linear peptide Modified-site 4 /product=“OTHER” /note= “Xaa = Ser or Thr” 46 Asn Xaa Xaa Xaa 1 736 amino acidsamino acid <Unknown> linear protein Protein 1..736 /note= “full lengthhuman small conductance, calcium-activated potassium channel protein 3(hSK3)” 47 Met Asp Thr Ser Gly His Phe His Asp Ser Gly Val Gly Asp LeuAsp 1 5 10 15 Glu Asp Pro Lys Cys Pro Cys Pro Ser Ser Gly Asp Glu GlnGln Gln 20 25 30 Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Pro Pro Pro AlaPro Pro 35 40 45 Ala Ala Pro Gln Gln Pro Leu Gly Pro Ser Leu Gln Pro GlnPro Pro 50 55 60 Gln Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln GlnGln Gln 65 70 75 80 Gln Gln Gln Gln Gln Pro Pro His Pro Leu Ser Gln LeuAla Gln Leu 85 90 95 Gln Ser Gln Pro Val His Pro Gly Leu Leu His Ser SerPro Thr Ala 100 105 110 Phe Arg Ala Pro Pro Ser Ser Asn Ser Thr Ala IleLeu His Pro Ser 115 120 125 Ser Arg Gln Gly Ser Gln Leu Asn Leu Asn AspHis Leu Leu Gly His 130 135 140 Ser Pro Ser Ser Thr Ala Thr Ser Gly ProGly Gly Gly Ser Arg His 145 150 155 160 Arg Gln Ala Ser Pro Leu Val HisArg Arg Asp Ser Asn Pro Ser Thr 165 170 175 Glu Ile Ala Met Ser Ser CysLys Tyr Ser Gly Gly Val Met Lys Pro 180 185 190 Leu Ser Arg Leu Ser AlaSer Arg Arg Asn Leu Ile Glu Ala Glu Thr 195 200 205 Glu Gly Gln Pro LeuGln Leu Phe Ser Pro Ser Asn Pro Pro Glu Ile 210 215 220 Val Ile Ser SerArg Glu Asp Asn His Ala His Gln Thr Leu Leu His 225 230 235 240 His ProAsn Ala Thr His Asn His Gln His Ala Gly Thr Thr Ala Ser 245 250 255 SerThr Thr Phe Pro Lys Ala Asn Lys Arg Lys Asn Gln Asn Ile Gly 260 265 270Tyr Lys Leu Gly His Arg Arg Ala Leu Phe Glu Lys Arg Lys Arg Leu 275 280285 Ser Asp Tyr Ala Leu Ile Phe Gly Met Phe Gly Ile Val Val Met Val 290295 300 Ile Glu Thr Glu Leu Ser Trp Gly Leu Tyr Ser Lys Asp Ser Met Phe305 310 315 320 Ser Leu Ala Leu Lys Cys Leu Ile Ser Leu Ser Thr Ile IleLeu Leu 325 330 335 Gly Leu Ile Ile Ala Tyr His Thr Arg Glu Val Gln LeuPhe Val Ile 340 345 350 Asp Asn Gly Ala Asp Asp Trp Arg Ile Ala Met ThrTyr Glu Arg Ile 355 360 365 Leu Tyr Ile Ser Leu Glu Met Leu Val Cys AlaIle His Pro Ile Pro 370 375 380 Gly Glu Tyr Lys Phe Phe Trp Thr Ala ArgLeu Ala Phe Ser Tyr Thr 385 390 395 400 Pro Ser Arg Ala Glu Ala Asp ValAsp Ile Ile Leu Ser Ile Pro Met 405 410 415 Phe Leu Arg Leu Tyr Leu IleAla Arg Val Met Leu Leu His Ser Lys 420 425 430 Leu Phe Thr Asp Ala SerSer Arg Ser Ile Gly Ala Leu Asn Lys Ile 435 440 445 Asn Phe Asn Thr ArgPhe Val Met Lys Thr Leu Met Thr Ile Cys Pro 450 455 460 Gly Thr Val LeuLeu Val Phe Ser Ile Ser Leu Trp Ile Ile Ala Ala 465 470 475 480 Trp ThrVal Arg Val Cys Glu Arg Tyr His Asp Gln Gln Asp Val Thr 485 490 495 SerAsn Phe Leu Gly Ala Met Trp Leu Ile Ser Ile Thr Phe Leu Ser 500 505 510Ile Gly Tyr Gly Asp Met Val Pro His Thr Tyr Cys Gly Lys Gly Val 515 520525 Cys Leu Leu Thr Gly Ile Met Gly Ala Gly Cys Thr Ala Leu Val Val 530535 540 Ala Val Val Ala Arg Lys Leu Glu Leu Thr Lys Ala Glu Lys His Val545 550 555 560 His Asn Phe Met Met Asp Thr Gln Leu Thr Lys Arg Ile LysAsn Ala 565 570 575 Ala Ala Asn Val Leu Arg Glu Thr Trp Leu Ile Tyr LysHis Thr Lys 580 585 590 Leu Leu Lys Lys Ile Asp His Ala Lys Val Arg LysHis Gln Arg Lys 595 600 605 Phe Leu Gln Ala Ile His Gln Leu Arg Ser ValLys Met Glu Gln Arg 610 615 620 Lys Leu Ser Asp Gln Ala Asn Thr Leu ValAsp Leu Ser Lys Met Gln 625 630 635 640 Asn Val Met Tyr Asp Leu Ile ThrGlu Leu Asn Asp Arg Ser Glu Asp 645 650 655 Leu Glu Lys Gln Ile Gly SerLeu Glu Ser Lys Leu Glu His Leu Thr 660 665 670 Ala Ser Phe Asn Ser LeuPro Leu Leu Ile Ala Asp Thr Leu Arg Gln 675 680 685 Gln Gln Gln Gln LeuLeu Ser Ala Ile Ile Glu Ala Arg Gly Val Ser 690 695 700 Val Ala Val GlyThr Thr His Thr Pro Ile Ser Asp Ser Pro Ile Gly 705 710 715 720 Val SerSer Thr Ser Phe Pro Thr Pro Tyr Thr Ser Ser Ser Ser Cys 725 730 735 2462base pairs nucleic acid single linear cDNA - 1..2462 /note= “human smallconductance, calcium-activated potassium channel protein 3 (hSK3) fulllength cDNA” 48 AGTTCTTTCA CCCCCTCTTC TTTCTCCAAG CTCCCCTCCT GCTCTCCCTCCCTGCCCAAT 60 ACAATGCATT CTTGAGTGGC AGCGTCTGGA CTCCAGGCAG CCCCAGAGAACCGAAGCAAG 120 CCAAAGAGAG GACTGGAGCC AAGATACTGG TGGGGGAGAT TGGATGCCTGGCTTTCTTTG 180 AGGACATCTT TGGAGCGAGG GTGGCTTTGG GGTGGGGGCT TGTGCTGCAGGGAATACAGC 240 CAGGCCCCAA GATGGACACT TCTGGGCACT TCCATGACTC GGGGGTGGGGGACTTGGATG 300 AAGACCCCAA GTGCCCCTGT CCATCCTCTG GGGATGAGCA GCAGCAGCAGCAGCAGCAGC 360 AACAGCAGCA GCAGCCACCA CCGCCAGCGC CACCAGCAGC CCCCCAGCAGCCCCTGGGAC 420 CCTCGCTGCA GCCTCAGCCT CCGCAGCTTC AGCAGCAGCA GCAGCAGCAGCAGCAGCAGC 480 AGCAGCAGCA GCAGCAGCAG CAGCAGCCAC CGCATCCCCT GTCTCAGCTCGCCCAACTCC 540 AGAGCCAGCC CGTCCACCCT GGCCTGCTGC ACTCCTCTCC CACCGCTTTCAGGGCCCCCC 600 CTTCGTCCAA CTCCACCGCC ATCCTCCACC CTTCCTCCAG GCAAGGCAGCCAGCTCAATC 660 TCAATGACCA CTTGCTTGGC CACTCTCCAA GTTCCACAGC TACAAGTGGGCCTGGCGGAG 720 GCAGCCGGCA CCGACAGGCC AGCCCCCTGG TGCACCGGCG GGACAGCAACCCCTCCACGG 780 AGATCGCCAT GAGCTCCTGC AAGTATAGCG GTGGGGTCAT GAAGCCCCTCAGCCGCCTCA 840 GCGCCTCCCG GAGGAACCTC ATCGAGGCCG AGACTGAGGG CCAACCCCTCCAGCTTTTCA 900 GCCCTAGCAA CCCCCCGGAG ATCGTCATCT CCTCCCGGGA GGACAACCATGCCCACCAGA 960 CCCTGCTCCA TCACCCTAAT GCCACCCACA ACCACCAGCA TGCCGGCACCACCGCCAGCA 1020 GCACCACCTT CCCCAAAGCC AACAAGCGGA AAAACCAAAA CATTGGCTATAAGCTGGGAC 1080 ACAGGAGGGC CCTGTTTGAA AAGAGAAAGC GACTGAGTGA CTATGCTCTGATTTTTGGGA 1140 TGTTTGGAAT TGTTGTTATG GTGATAGAGA CCGAGCTCTC TTGGGGTTTGTACTCAAAGG 1200 ACTCCATGTT TTCGTTGGCC CTGAAATGCC TTATCAGTCT GTCCACCATCATCCTTTTGG 1260 GCTTGATCAT CGCCTACCAC ACACGTGAAG TCCAGCTCTT CGTGATCGACAACGGCGCGG 1320 ATGACTGGCG GATAGCCATG ACCTACGAGC GCATCCTCTA CATCAGCCTGGAGATGCTGG 1380 TGTGCGCCAT CCACCCCATT CCTGGCGAGT ACAAGTTCTT CTGGACGGCACGCCTGGCCT 1440 TCTCCTACAC ACCCTCCCGG GCGGAGGCCG ATGTGGACAT CATCCTGTCTATCCCCATGT 1500 TCCTGCGCCT GTACCTGATC GCCCGAGTCA TGCTGCTGCA CAGCAAGCTCTTCACCGATG 1560 CCTCGTCCCG CAGCATCGGG GCCCTCAACA AGATCAACTT CAACACCCGCTTTGTCATGA 1620 AGACGCTCAT GACCATCTGC CCTGGCACTG TGCTGCTCGT GTTCAGCATCTCTCTGTGGA 1680 TCATTGCTGC CTGGACCGTC CGTGTCTGTG AAAGGTACCA TGACCAGCAGGACGTAACTA 1740 GTAACTTTCT GGGTGCCATG TGGCTCATCT CCATCACATT CCTTTCCATTGGTTATGGGG 1800 ACATGGTGCC CCACACATAC TGTGGGAAAG GTGTCTGTCT CCTCACTGGCATCATGGGTG 1860 CAGGCTGCAC TGCCCTTGTG GTGGCCGTGG TGGCCCGAAA GCTGGAACTCACCAAAGCGG 1920 AGAAGCACGT TCATAACTTC ATGATGGACA CTCAGCTCAC CAAGCGGATCAAGAATGCTG 1980 CAGCCAATGT CCTTCGGGAA ACATGGTTAA TCTATAAACA CACAAAGCTGCTAAAGAAGA 2040 TTGACCATGC CAAAGTGAGG AAACACCAGA GGAAGTTCCT CCAAGCTATCCACCAGTTGA 2100 GGAGCGTCAA GATGGAACAG AGGAAGCTGA GTGACCAAGC CAACACTCTGGTGGACCTTT 2160 CCAAGATGCA GAATGTCATG TATGACTTAA TCACAGAACT CAATGACCGGAGCGAAGACC 2220 TGGAGAAGCA GATTGGCAGC CTGGAGTCGA AGCTGGAGCA TCTCACCGCCAGCTTCAACT 2280 CCCTGCCGCT GCTCATCGCC GACACCCTGC GCCAGCAGCA GCAGCAGCTCCTGTCTGCCA 2340 TCATCGAGGC CCGGGGTGTC AGCGTGGCAG TGGGCACCAC CCACACCCCAATCTCCGATA 2400 GCCCCATTGG GGTCAGCTCC ACCTCCTTCC CGACCCCGTA CACAAGTTCAAGCAGTTGCT 2460 AA 2462

What is claimed is:
 1. An isolated nucleic acid encoding a monomer of acalcium-activated potassium channel, said monomer: i. having acalculated molecular weight of between 40 and 80 kDa; ii. having a unitconductance of between 2 and 60 pS when the monomer is in the functionalpolymeric form of a potassium channel and is expressed in a Xenopusoocyte; and; iii. specifically binding to antibodies generated againstSEQ ID NOS:30 or
 42. 2. An isolated nucleic acid encoding at least 15contiguous amino acids from a calcium-activated potassium channelprotein, said protein having a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, SEQ ID NO:47 andconservatively modified variants thereof, with the provisio that thecontiguous amino acids do not consist of a glutamine repeat amino acidsequence.
 3. The isolated nucleic acid of claim 2 wherein said nucleicacid encodes a calcium activated potassium channel protein having aconductance of between 2 and 60 pS and a molecular weight of between 40and 80 kilodaltons and wherein said nucleic acid either i. selectivelyhybridizes under moderate stringency hybridization conditions to asequence selected from the group consisting of SEQ ID NOS:13, 14, 15,16, 21, 22, 31, 44, and 48, or ii. encodes a protein which could beencoded by a nucleic acid that selectively hybridizes under moderatestringency hybridization conditions to a sequence selected from thegroup consisting of SEQ ID NOS:13, 14, 15, 16, 21, 22, 31, 44, and 48.4. The isolated nucleic acid of claim 1, wherein said nucleic acidencodes a protein having a sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, and SEQ IDNO:47.
 5. The isolated nucleic acid of claim 1, wherein said nucleicacid encodes a protein having a sequence selected from the groupconsisting 3f SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:43.6. An isolated nucleic acid of claim 1 wherein the sequence is identicalto a naturally occurring sequence.
 7. An isolated nucleic acid of claim1 having the sequence depicted in SEQ ID NO:31.
 8. An isolated nucleicacid of claim 1 encoding at least 15 contiguous amino acids of a monomerof an intermediate conductance, calcium-activated potassium channel,said monomer: i. having a calculated molecular weight of about 42 to 52kDa; ii. having units conductance of between 30 and 60 pS in the inwarddirection when the monomer is in the functional polymeric form of apotassium channel and is expressed in a Xenopus oocyte; and, iii.specifically binding to a polyclonal antibody generated against SEQ IDNO:32.
 9. An isolated nucleic acid of claim 8 wherein the sequence isidentical to a naturally occurring sequence.
 10. An isolated nucleicacid of claim 6 encoding any 8 contiguous amino acids from the followingsequence: VRGPPCVQDLGAPLTSPQPWPGFLGQGEAL (SEQ ID NO:33).
 11. An isolatednucleic acid sequence of at least 20 nucleotides in length whichspecifically hybridizes, under stringent conditions, to a nucleic acidencoding an intermediate calcium-activated potassium channel protein,said protein selected from the group consisting of SEQ ID NO:32.
 12. Anisolated calcium-activated potassium channel protein having at least 15contiguous amino acids from a sequence selected from the groupconsisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, SEQ ID NO:47 andconservatively modified variants thereof, wherein said variantsspecifically react, under immunologically reactive conditions, with anantibody reactive to a protein selected from the group consisting of:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47.
 13. The isolatedcalcium-activated potassium channel protein of claim 12, wherein saidprotein when expressed in a Xenopus oocyte leads to formation of ancalcium-activated potassium channel having a conductance of between 2and 80 pS and a molecular weight of between 40 and 80 kilodaltons. 14.The isolated calcium-activated potassium channel protein of claim 12,wherein said protein has a sequence shown in SEQ ID NO:1, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:32 or SEQ ID NO:47.
 15. The isolatedcalcium-activated potassium channel protein of claim 12, wherein saidprotein has a sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:43.
 16. An isolatedprotein of claim 12 comprising at least 15 contiguous amino acids from amonomer of a calcium-activated potassium channel protein having i.having a calculated molecular weight of about 42 to about 52 kDa; ii.having units conductance of between 30 and 60 pS in the inward directionwhen the monomer is in the functional polymeric form of a potassiumchannel and is expressed in a Xenopus oocyte; and; iii. specificallybinding to a polyclonal antibody generated against SEQ ID NO:32.
 17. Anisolated protein of claim 16 having an amino acid sequence identical toa naturally occurring sequence.
 18. An isolated protein of claim 16having the sequence depicted in SEQ ID NO:32.
 19. An isolated nucleicacid of claim 16 encoding any 8 contiguous amino acids from thefollowing sequence: VRGPPCVQDLGAPLTSPQPWPGFLGQGEAL (SEQ ID NO:33). 20.An isolated intermediate conductance calcium-activated potassium channelprotein encoded by a nucleic acid a portion of which when amplified byprimer pairs produces an amplified fragment which selectivelyhybridizes, under stringent hybridization conditions to SEQ ID NO:31wherein said primer pairs are selected from the group consisting of: 5′GCCGTGCGTGCAGGATTTAGG 3′ (SEQ ID NO:34)5′ CCAGAGGCCAAGCGTGAGGCC 3′ (SEQID NO:35);5′ TCCAAGATGCACATGATCCTG 3′ (SEQ ID NO:36); and,5′GGACTGCTGGCTGGGTTCTGG 3′ (SEQ ID NO:37).
 21. An antibody specificallyreactive, under immunologically reactive conditions, to acalcium-activated potassium channel protein, said protein having asequence selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47.
 22. An antibody of claim 21,wherein said antibody is specifically reactive to the protein selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:32, and SEQ ID NO:47.
 23. An antibody of claim 21, whereinsaid protein has a sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:43.
 24. The antibody ofclaim 21, wherein said antibody is a monoclonal antibody.
 25. Theantibody of claim 24, wherein said monoclonal antibody is specificallyreactive to a protein selected from the group consisting of SEQ ID NO:1,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, and SEQ ID NO:47.
 26. Anexpression vector comprising a nucleic acid encoding a calcium-activatedpotassium channel protein, said channel protein having a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ IDNO:47, and conservatively modified variants thereof, wherein saidconservatively modified variant is a protein which when expressed in anoocyte leads to formation of a calcium-activated potassium channelhaving a conductance of between 2 and 80 pS, a molecular weight ofbetween 40 and 80 kilodaltons, and specifically reacts, underimmunologically reactive conditions, with an antibody reactive to thechannel protein selected from the group consisting of: SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47.
 27. A host cell transfected withthe vector of claim
 26. 28. An isolated nucleic acid sequence of atleast 20 nucleotides in length which specifically hybridizes, understringent conditions, to a nucleic acid encoding a calcium-activatedpotassium channel protein, said protein selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47.
 29. Amethod for detecting the presence of a calcium-activated potassiumchannel protein in a biological sample, said method comprising: (a)contacting said biological sample with an antibody, wherein saidantibody specifically reacts, under immunologically reactive conditions,to the channel protein having a sequence selected from the groupconsisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47;(b) allowing said antibody to bind to said protein under immunologicallyreactive conditions, wherein detection of said bound antibody indicatesthe presence of said channel protein.
 30. A method for detecting thepresence, in a biological sample, of a nucleic acid sequence encoding acalcium-activated potassium channel protein of at least 25 amino acidsin length, said method comprising: (a) contacting said sample, understringent hybridization conditions, with a nucleic acid probe comprisinga nucleic acid segment that selectively hybridizes to a nucleic acidencoding said channel protein having a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47; (b)allowing said nucleic acid encoding the channel protein to selectivelyhybridize to said probe to form a hybridization complex, whereindetection of said hybridization complex is an indication of the presenceof said nucleic acid sequence in said sample.
 31. An isolatedcalcium-activated potassium channel protein encoded by a nucleic acidamplified by primers which selectively hybridize, under stringenthybridization conditions, to the same sequence as primers selected fromthe group consisting of: for hSK1 ATGCCGGGTCCCCGGGCGGCCTGC (SEQ IDNO:5)TCACCCGCAGTCCGAGGAGGCCAC (SEQ ID NO:6) for rSK2ATGAGCAGCTGCAGGTACAACGGG (SEQ ID NO:7)CTAGCTACTCTCAGATGAAGTTGG (SEQ IDNO:8) for rSK3 ATGAGCTCCTGCAAATACAGCGGT (SEQ IDNO:9)TTAGCAACTCTGTGAACTTG (SEQ ID NO:10) for rSK1TCAGGGAAGCCCCCGACCGTCAGT (SEQ ID NO:11)TCACCCACAGTCTGATGCCGTGGT (SEQ IDNO:12) for hSK2 ATGAGCAGCTGCAGGTACAACG (SEQ IDNO:23)CTAGCTACTCTCTGATGAAGTTG (SEQ ID NO:24) for hSK3ATGAGCTCCTGCAAGTATAGC (SEQ ID NO:25)TTAGCAACTGCTTGAACTTGTG (SEQ IDNO:26) for hIK1 GCCGTGCGTGCAGGATTTAGG (SEQ IDNO:34)CCAGAGGCCAAGCGTGAGGCC (SEQ ID NO:35); and, for hIK1TCCAAGATGCACATGATCCTG (SEQ ID NO:36)GGACTGCTGGCTGGGTTCTGG (SEQ IDNO:37).
 32. A method of identifying a compound which increases ordecreases the potassium ion flux through a calcium-activated potassiumchannel, the steps of: a) contacting said compound with a eukaryotichost cell or cell membrane in which has been expressed acalcium-activated potassium channel protein having a sequence selectedfrom the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQID NO:47 conservatively modified variants thereof, wherein saidconservatively modified variants specifically bind to antibodiesspecifically reactive with an antigen having an amino acid sequenceselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ IDNO:43, and SEQ ID NO:47, have a conductance of between 2 and 80 pS, anda molecular weight between 40 and 80 kilodaltons; and b) determining thefunctional effect of the compound upon the cell or cell membraneexpressing said channel.
 33. The method of claim 32, wherein theincreased or decreased flux of potassium ions is determined by measuringthe electrical current across the cell membrane of said eukaryotic hostcell.
 34. The method of claim 32, where said channel protein has asequence selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47.
 35. The method of claim 32,wherein said channel protein is recombinant.
 36. The isolated nucleicacid of claim 1, said nucleic acid having a sequence selected from thegroup consisting of: SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:31, SEQ ID NO:44, and SEQID NO:48.
 37. An isolated eukaryotic nucleic acid encoding acalcium-activated channel protein of at least 400 amino acid residues inlength, wherein said channel protein comprises an amino acid sequencehaving at least 60% similarity over at least the length of a core regionof a protein selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:32, SEQ ID NO:43, and SEQ ID NO:47, and wherein said channel proteinhas a conductance of between 2 and 80 pS.
 38. The isolated nucleic acidof claim 37, wherein said channel protein has at least 90% sequencesimilarity over a comparison window of at least 20 contiguous amino acidresidues within said core region.
 39. A vector comprising the isolatednucleic acid of claim
 37. 40. A host cell transfected with the vector ofclaim
 39. 41. A method of making a calcium-activated potassium channelprotein, comprising culturing the host cell of claim 40 under conditionspermitting expression of said nucleic acid encoding said channelprotein.
 42. A method of making a calcium-activated potassium channelprotein, comprising culturing the host cell of claim 27 under conditionspermitting expression of said nucleic acid encoding said channelprotein.
 43. A method of identifying a compound that increases ordecreases the potassium ion flux through a calcium-activated potassiumchannel, the steps of: a) contacting said compound with a host cell orcell membrane in which has been expressed a calcium-activated potassiumchannel protein of at least 400 amino acid residues in length, whereinsaid SK channel protein has an amino acid sequence having at least 60%similarity over the length of the core region of a protein selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ IDNO:47, and wherein said channel protein has a conductance of between 2and 80 pS; and b) determining the functional effect of the compound uponthe cell or cell membrane expressing the channel.
 44. The method ofclaim 30, wherein said stringent hybridization conditions are moderatestringency hybridization conditions.
 45. The method of claim 44, whereinsaid nucleic acid sequence encodes a calcium-activated potassium channelprotein of at least 400 amino acid residues in length and having aconductance of between 2 and 80 pS.
 46. The method of claim 45, whereinsaid nucleic acid probe comprises at least 250 contiguous nucleotidesencoding a subsequence within said channel protein core region.
 47. Theprotein encoded by the isolated nucleic acid of claim
 37. 48. The methodof claim 43, wherein the increased or decreased flux of potassium ionsis determined by measuring the electrical current across the cellmembrane of said eukaryotic host cell.
 49. In a computer system, amethod of screening for mutations of SK and IK genes, the methodcomprising the steps of: (i) receiving input of a first nucleic acidsequence encoding a calcium-activated channel protein having a sequenceselected from the group consisting of SEQ ID NOS:1, 2, 3, 4, 19, 20, 32,43, 47 and conservatively modified versions thereof; (ii) comparing thefirst nucleic acid sequence with a second nucleic acid sequence havingsubstantial identity to the first nucleic acid sequence; and (iii)identifying nucleotide differences between the first and second nucleicacid sequences.
 50. The method of claim 49, wherein the second nucleicacid sequence is associated with a disease state.
 51. In a computersystem, a method for identifying a three-dimensional structure of SK andIK proteins, the method comprising the steps of: (i) receiving input ofan amino acid sequence of a calcium-activated channel protein or anucleotide sequence of a gene encoding the protein, the protein havingan amino acid sequence selected from the group consisting of SEQ IDNOS:1, 2, 3, 4, 19, 20, 32, 43, 47, and conservatively modified versionsthereof; and (ii) generating a three-dimensional structure of theprotein encoded by the amino acid sequence.
 52. The method of claim 51,wherein said amino acid sequence is a primary structure and wherein saidgenerating step includes the steps of forming a secondary structure fromsaid primary structure using energy terms encoded by the primarystructure and forming a tertiary structure from said secondary structureusing energy terms encoded by said secondary structure.
 53. The methodof claim 51, wherein said generating step includes the step of forming aquaternary structure from said tertiary structure using anisotropy termsencoded by the tertiary structure.
 54. The method of claim 52, whereinsaid generating step further includes the step of forming a quaternarystructure from said tertiary structure using anisotropy terms encoded bythe tertiary structure.
 55. The method of claim 50, further comprisingthe step of identifying regions of the three-dimensional structure ofthe protein that bind to ligands and using the regions to identifyligands that bind to the protein.